High throughput screening assay systems in microscale fluidic devices

ABSTRACT

The present invention provides novel microfluidic devices and methods that are useful for performing high-throughput screening assays. In particular, the devices and methods of the invention are useful in screening large numbers of different compounds for their effects on a variety of chemical, and preferably, biochemical systems.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This is a continuation-in-part of U.S. patent application Ser.No. 08/671,987 filed Jun. 28, 1996, and U.S. patent application Ser. No.08/761,575 filed Dec. 6, 1996, each of which is hereby incorporatedherein by reference in its entirety for all purposes. A PCT Applicationdesignating the United States of America, Attorney Docket No.017646-00042PC, substantially identical to the present application, wasco-filed in the United States Receiving Office on Jun. 24, 1997. Thisapplication is also incorporated herein by reference.

FIELD OF THE INVENTION

[0002] This application relates to apparatus and assay systems fordetecting molecular interactions. The apparatus comprise a substratewith one or more intersecting channels and an electroosmotic fluidmovement component, or other component for moving fluid in the channelson the substrate.

BACKGROUND OF THE INVENTION

[0003] There has long been a need for the ability to rapidly assaycompounds for their effects on various biological processes. Forexample, enzymologists have long sought better substrates, betterinhibitors or better catalysts for enzymatic reactions. Similarly, inthe pharmaceutical industries, attention has been focused on identifyingcompounds that may block, reduce, or even enhance the interactionsbetween biological molecules. Specifically, in biological systems theinteraction between a receptor and its ligand often may result, eitherdirectly or through some downstream event, in either a deleterious orbeneficial effect on that system, and consequently, on a patient forwhom treatment is sought. Accordingly, researchers have long soughtafter compounds or mixtures of compounds that can reduce, block or evenenhance that interaction. Similarly, the ability to rapidly processsamples for detection of biological molecules relevant to diagnostic orforensic analysis is of fundamental value for, e.g., diagnosticmedicine, archaeology, anthropology, and modern criminal investigation.

[0004] Modem drug discovery is limited by the throughput of the assaysthat are used to screen compounds that possess these described effects.In particular, screening of a maximum number of different compoundsnecessitates reducing the time and labor requirements associated witheach screen.

[0005] High throughput screening of collections of chemicallysynthesized molecules and of natural products (such as microbialfermentation broths) has thus played a central role in the search forlead compounds for the development of new pharmacological agents. Theremarkable surge of interest in combinatorial chemistry and theassociated technologies for generating and evaluating moleculardiversity represent significant milestones in the evolution of thisparadigm of drug discovery. See Pavia et al., 1993, Bioorg. Med. Chem.Lett. 3: 387-396, incorporated herein by reference. To date, peptidechemistry has been the principle vehicle for exploring the utility ofcombinatorial methods in ligand identification. See Jung &Beck-Sickinger, 1992, Angew. Chem. Int. Ed. Engl. 31: 367-383,incorporated herein by reference. This may be ascribed to theavailability of a large and structurally diverse range of amino acidmonomers, a relatively generic, high-yielding solid phase couplingchemistry and the synergy with biological approaches for generatingrecombinant peptide libraries. Moreover, the potent and specificbiological activities of many low molecular weight peptides make thesemolecules attractive starting points for therapeutic drug discovery. SeeHirschmann, 1991, Angew. Chem. Int. Ed. Engl. 30: 1278-1301, and Wiley &Rich, 1993, Med. Res. Rev. 13: 327-384, each of which is incorporatedherein by reference. Unfavorable pharmacodynamic properties such as poororal bioavailability and rapid clearance in vivo have limited the morewidespread development of peptidic compounds as drugs, however. Thisrealization has recently inspired workers to extend the concepts ofcombinatorial organic synthesis beyond peptide chemistry to createlibraries of known pharmacophores like benzodiazepines (see Bunin &Ellman, 1992, J. Amer. Chem. Soc. 114: 10997-10998, incorporated hereinby reference) as well as polymeric molecules such as oligomericN-substituted glycines (“peptoids”) and oligocarbamates. See Simon etal., 1992, Proc. Natl. Acad. Sci. USA 89: 9367-9371; Zuckermann et al.,1992, J. Amer. Chem. Soc. 114: 10646-10647; and Cho et al., 1993,Science 261:1303-1305, each of which is incorporated herein byreference.

[0006] In similar developments, much as modern combinatorial chemistryhas resulted in a dramatic increase in the number of test compounds thatmay be screened, human genome research has also uncovered large numbersof new target molecules (e.g., genes and gene products such as proteinsand RNA) against which the efficacy of test compounds are screened.

[0007] Despite the improvements achieved using parallel screeningmethods and other technological advances, such as robotics and highthroughput detection systems, current screening methods still have anumber of associated problems. For example, screening large numbers ofsamples using existing parallel screening methods have high spacerequirements to accommodate the samples and equipment, e.g., robotics,etc., high costs associated with that equipment, and high reagentrequirements necessary for performing the assays. Additionally, in manycases, reaction volumes must be very small to account for the smallamounts of the test compounds that are available. Such small volumescompound errors associated with fluid handling and measurement, e.g.,due to evaporation, small dispensing errors, or the like. Additionally,fluid handling equipment and methods have typically been unable tohandle these volume ranges with any acceptable level of accuracy due inpart to surface tension effects in such small volumes.

[0008] The development of systems to address these problems mustconsider a variety of aspects of the assay process. Such aspects includetarget and compound sources, test compound and target handling, specificassay requirements, and data acquisition, reduction storage andanalysis. In particular, there exists a need for high throughputscreening methods and associated equipment and devices that are capableof performing repeated, accurate assay screens, and operating at verysmall volumes.

[0009] The present invention meets these and a variety of other needs.In particular, the present invention provides novel methods andapparatuses for performing screening assays which address and providemeaningful solutions to these problems.

SUMMARY OF THE INVENTION

[0010] The present invention provides methods of screening a pluralityof test compounds for an effect on a biochemical system. These methodstypically utilize microfabricated substrates which have at least a firstsurface, and at least two intersecting channels fabricated into thatfirst surface. At least one of the intersecting channels will have atleast one cross-sectional dimension in a range from 0.1 to 500 μm. Themethods involve flowing a first component of a biochemical system in afirst of the at least two intersecting channels. At least a first testcompound is flowed from a second channel into the first channel wherebythe test compound contacts the first component of the biochemicalsystem. An effect of the test compound on the biochemical system is thendetected.

[0011] In a related aspect, the method comprises continuously flowingthe first component of a biochemical system in the first channel of theat least two intersecting channels. Different test compounds areperiodically introduced into the first channel from a second channel.The effect, if any, of the test compound on the biochemical system isthen detected.

[0012] In an alternative aspect, the methods utilize a substrate havingat least a first surface with a plurality of reaction channelsfabricated into the first surface. Each of the plurality of reactionchannels is fluidly connected to at least two transverse channels alsofabricated in the surface. The at least first component of a biochemicalsystem is introduced into the plurality of reaction channels, and aplurality of different test compounds is flowed through at least one ofthe at least two transverse channels. Further, each of the plurality oftest compounds is introduced into the transverse channel in a discretevolume. Each of the plurality of different test compounds is directedinto a separate reaction channel and the effect of each of the testcompounds on the biochemical system is then detected.

[0013] The present invention also provides apparatuses for practicingthe above methods. In one aspect, the present invention provides anapparatus for screening test compounds for an effect on a biochemicalsystem. The device comprises a substrate having at least one surfacewith at least two intersecting channels fabricated into the surface. Theat least two intersecting channels have at least one cross-sectionaldimension in the range from about 0.1 to about 500 μm. The device alsocomprises a source of different test compounds fluidly connected to afirst of the at least two intersecting channels, and a source of atleast one component of the biochemical system fluidly connected to asecond of the at least two intersecting channels. Also included arefluid direction systems for flowing the at least one component withinthe intersecting channels, and for introducing the different testcompounds from the first to the second of the intersecting channels. Theapparatus also optionally comprises a detection zone in the secondchannel for detecting an effect of said test compound on saidbiochemical system.

[0014] In preferred aspects, the apparatus of the invention includes afluid direction system which comprises at least three electrodes, eachelectrode being in electrical contact with the at least two intersectingchannels on a different side of an intersection formed by the at leasttwo intersecting channels. The fluid direction system also includes acontrol system for concomitantly applying a variable voltage at each ofthe electrodes, whereby movement of the test compounds or the at leastfirst component in the at least two intersecting channels arecontrolled.

[0015] In another aspect, the present invention provides an apparatusfor detecting an effect of a test compound on a biochemical system,comprising a substrate having at least one surface with a plurality ofreaction channels fabricated into the surface. The apparatus also has atleast two transverse channels fabricated into the surface, wherein eachof the plurality of reaction channels is fluidly connected to a first ofthe at least two transverse channels at a first point in each of thereaction channels, and fluidly connected to a second transverse channelat a second point in each of the reaction channels. The apparatusfurther includes a source of at least one component of the biochemicalsystem fluidly connected to each of the reaction channels, a source oftest compounds fluidly connected to the first of the transversechannels, and a fluid direction system for controlling movement of thetest compound and the first component within the transverse channels andthe plurality of reaction channels. As above, the apparatuses alsooptionally include a detection zone in the second transverse channel fordetecting an effect of the test compound on the biochemical system.

BRIEF DESCRIPTION OF THE DRAWING

[0016]FIG. 1 is a schematic illustration of one embodiment of amicrolaboratory screening assay system of the present invention whichcan be used in running a continuous flow assay system.

[0017]FIGS. 2A and 2B show a schematic illustration of the apparatusshown in FIG. 1, operating in alternate assay systems. FIG. 2A shows asystem used for screening effectors of an enzyme-substrate interaction.FIG. 2B illustrates the use of the apparatus in screening effectors ofreceptor-ligand interactions.

[0018]FIG. 3 is a schematic illustration of a “serial input parallelreaction” microlaboratory assay system in which compounds to be screenedare serially introduced into the device but then screened in a parallelorientation within the device.

[0019] FIGS. 4A-4F show a schematic illustration of the operation of thedevice shown in FIG. 3, in screening a plurality of bead based testcompounds.

[0020]FIG. 5 shows a schematic illustration of a continuous flow assaydevice incorporating a sample shunt for performing prolonged incubationfollowed by a separation step.

[0021]FIG. 6A shows a schematic illustration of a serial input parallelreaction device for use with fluid based test compounds. FIGS. 6B and 6Cshow a schematic illustration of fluid flow patterns within the deviceshown in FIG. 6A.

[0022]FIG. 7 shows a schematic illustration of one embodiment of anoverall assay systems which employs multiple microlaboratory deviceslabeled as “LabChips™” for screening test compounds.

[0023]FIG. 8 is a schematic illustration of a chip layout used for acontinuous-flow assay screening system.

[0024]FIG. 9 shows fluorescence data from a continuous flow assayscreen. FIG. 9A shows fluorescence data from a test screen whichperiodically introduced a known inhibitor (IPTG) into a β-galactosidaseassay system in a chip format. FIG. 9B shows a superposition of two datasegments from FIG. 9A, directly comparing the inhibitor data withcontrol (buffer) data.

[0025]FIG. 10 illustrates the operating parameters of a fluid flowsystem on a small chip device for performing enzyme inhibitor screening.

[0026]FIG. 11 shows a schematic illustration of timing for sample/spacerloading in a microfluidic device channel.

[0027]FIG. 12, panels A-G schematically illustrate electrodes used inapparatuses of the invention.

DESCRIPTION OF THE PREFERRED EMBODIMENT

[0028] I. Applications for the Invention

[0029] The present invention provides novel microlaboratory systems andmethods that are useful for performing high-throughput screening assays.In particular, the present invention provides microfluidic devices andmethods of using such devices in screening large numbers of differentcompounds for their effects on a variety of chemical, and preferably,biochemical systems.

[0030] As used herein, the phrase “biochemical system” generally refersto a chemical interaction that involves molecules of the type generallyfound within living organisms. Such interactions include the full rangeof catabolic and anabolic reactions which occur in living systemsincluding enzymatic, binding, signalling and other reactions. Further,biochemical systems, as defined herein, also include model systems whichare mimetic of a particular biochemical interaction. Examples ofbiochemical systems of particular interest in practicing the presentinvention include, e.g., receptor-ligand interactions, enzyme-substrateinteractions, cellular signaling pathways, transport reactions involvingmodel barrier systems (e.g., cells or membrane fractions) forbioavailability screening, and a variety of other general systems.Cellular or organismal viability or activity may also be screened usingthe methods and apparatuses of the present invention, e.g., intoxicology studies. Biological materials which are assayed include, butare not limited to, cells, cellular fractions (membranes, cytosolpreparations, etc.), agonists and antagonists of cell membrane receptors(e.g., cell receptor-ligand interactions such as e.g., transferrin,c-kit, viral receptor ligands (e.g., CD4-HIV), cytokine receptors,chemokine receptors, interleukin receptors, immunoglobulin receptors andantibodies, the cadherein family, the integrin family, the selectinfamily, and the like; see, e.g., Pigott and Power (1993) The AdhesionMolecule FactsBook Academic Press New York and Hulme (ed) ReceptorLigand Interactions A Practical Approach Rickwood and Hames (serieseditors) IRL Press at Oxford Press NY), toxins and venoms, viralepitopes, hormones (e.g., opiates, steroids, etc.), intracellularreceptors (e.g. which mediate the effects of various small ligands,including steroids, thyroid hormone, retinoids and vitamin D; forreviews see, e.g., Evans (1988) Science, 240:889-895; Ham and Parker(1989) Curr. Opin. Cell Biol., 1:503-511; Burnstein et al. (1989), Ann.Rev. Physiol., 51:683-699; Truss and Beato (1993) Endocr. Rev.,14:459-479), peptides, retro-inverso peptides, polymers of α-, β-, orω-amino acids (D- or L-), enzymes, enzyme substrates, cofactors, drugs,lectins, sugars, nucleic acids (both linear and cyclic polymerconfigurations), oligosaccharides, proteins, phospholipids andantibodies. Synthetic polymers such as heteropolymers in which a knowndrug is covalently bound to any of the above, such as polyurethanes,polyesters, polycarbonates, polyureas, polyamides, polyethyleneimines,polyarylene sulfides, polysiloxanes, polyimides, and polyacetates arealso assayed. Other polymers are also assayed using the systemsdescribed herein, as would be apparent to one of skill upon review ofthis disclosure. One of skill will be generally familiar with thebiological literature. For a general introduction to biological systems,see, Berger and Kimmel, Guide to Molecular Cloning Techniques, Methodsin Enzymology volume 152 Academic Press, Inc., San Diego, Calif.(Berger); Sambrook et al. (1989) Molecular Cloning—A Laboratory Manual(2nd ed.) Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring HarborPress, NY, (Sambrook); Current Protocols in Molecular Biology, F. M.Ausubel et al., eds., Current Protocols, a joint venture between GreenePublishing Associates, Inc. and John Wiley & Sons, Inc., (through 1997Supplement) (Ausubel); Watson et al. (1987) Molecular Biology of theGene, Fourth Edition The Benjamin/Cummings Publishing Co., Menlo Park,Calif.; Watson et al. (1992) Recombinant DNA Second Edition ScientificAmerican Books, NY; Alberts et al. (1989) Molecular Biology of the CellSecond Edition Garland Publishing, NY; Pattison (1994) Principles andPractice of Clinical Virology; Darnell et al., (1990) Molecular CellBiology second edition, Scientific American Books, W. H. Freeman andCompany; Berkow (ed.) The Merck Manual of Diagnosis and Therapy, Merck &Co., Rahway, N J; Harrison's Principles of Internal Medicine, ThirteenthEdition, Isselbacher et al. (eds). (1994) Lewin Genes, 5th Ed., OxfordUniversity Press (1994); The “Practical Approach” Series of Books(Rickwood and Hames (series eds.) by IRL Press at Oxford UniversityPress, NY; The “FactsBook Series” of books from Academic Press, NY,;Product information from manufacturers of biological reagents andexperimental equipment also provide information useful in assayingbiological systems. Such manufacturers include, e.g., the SIGMA chemicalcompany (Saint Louis, Mo.), R&D systems (Minneapolis, Minn.), PharmaciaLKB Biotechnology (Piscataway, N.J.), CLONTECH Laboratories, Inc. (PaloAlto, Calif.), Chem Genes Corp., Aldrich Chemical Company (Milwaukee,Wis.), Glen Research, Inc., GIBCO BRL Life Technologies, Inc.(Gaithersberg, Md.), Fluka Chemica-Biochemika Analytika (Fluka ChemieAG, Buchs, Switzerland), Invitrogen, San Diego, Calif., and AppliedBiosystems (Foster City, Calif.), as well as many other commercialsources known to one of skill.

[0031] In order to provide methods and devices for screening compoundsfor effects on biochemical systems, the present invention generallyincorporates model in vitro systems which mimic a given biochemicalsystem in vivo for which effector compounds are desired. The range ofsystems against which compounds can be screened and for which effectorcompounds are desired, is extensive. For example, compounds areoptionally screened for effects in blocking, slowing or otherwiseinhibiting key events associated with biochemical systems whose effectis undesirable. For example, test compounds are optionally screened fortheir ability to block systems that are responsible, at least in part,for the onset of disease or for the occurrence of particular symptoms ofdiseases, including, e.g., hereditary diseases, cancer, bacterial orviral infections and the like. Compounds which show promising results inthese screening assay methods can then be subjected to further testingto identify effective pharmacological agents for the treatment ofdisease or symptoms of a disease.

[0032] Alternatively, compounds can be screened for their ability tostimulate, enhance or otherwise induce biochemical systems whosefunction is believed to be desirable, e.g., to remedy existingdeficiencies in a patient.

[0033] Once a model system is selected, batteries of test compounds canthen be applied against these model systems. By identifying those testcompounds that have an effect on the particular biochemical system, invitro, one can identify potential effectors of that system, in vivo.

[0034] In their simplest forms, the biochemical system models employedin the methods and apparatuses of the present invention will screen foran effect of a test compound on an interaction between two components ofa biochemical system, e.g., receptor-ligand interaction,enzyme-substrate interaction, and the like. In this form, thebiochemical system model will typically include the two normallyinteracting components of the system for which an effector is sought,e.g., the receptor and its ligand or the enzyme and its substrate.

[0035] Determining whether a test compound has an effect on thisinteraction then involves contacting the system with the test compoundand assaying for the functioning of the system, e.g., receptor-ligandbinding or substrate turnover. The assayed function is then compared toa control, e.g., the same reaction in the absence of the test compoundor in the presence of a known effector. Typically, such assays involvethe measurement of a parameter of the biochemical system. By “parameterof the biochemical system” is meant some measurable evidence of thesystem's functioning, e.g., the presence or absence of a labeled groupor a change in molecular weight (e.g., in binding reactions, transportscreens), the presence or absence of a reaction product or substrate (insubstrate turnover measurements), or an alteration in electrophoreticmobility (typically detected by a change in elution time of a labeledcompound).

[0036] Although described in terms of two-component biochemical systems,the methods and apparatuses may also be used to screen for effectors ofmuch more complex systems, where the result or end product of the systemis known and assayable at some level, e.g., enzymatic pathways, cellsignaling pathways and the like. Alternatively, the methods andapparatuses described herein are optionally used to screen for compoundsthat interact with a single component of a biochemical system, e.g.,compounds that specifically bind to a particular biochemical compound,e.g., a receptor, ligand, enzyme, nucleic acid, structuralmacromolecule, etc.

[0037] Biochemical system models may also be embodied in whole cellsystems. For example, where one is seeking to screen test compounds foran effect on a cellular response, whole cells are optionally utilized.Modified cell systems may also be employed in the screening systemsencompassed herein. For example, chimeric reporter systems areoptionally employed as indicators of an effect of a test compound on aparticular biochemical system. Chimeric reporter systems typicallyincorporate a heterogenous reporter system integrated into a signalingpathway which signals the binding of a receptor to its ligand. Forexample, a receptor is fused to a heterologous protein, e.g., an enzymewhose activity is readily assayable. Activation of the receptor byligand binding then activates the heterologous protein which then allowsfor detection. Thus, the surrogate reporter system produces an event orsignal which is readily detectable, thereby providing an assay forreceptor/ligand binding. Examples of such chimeric reporter systems havebeen previously described in the art.

[0038] Additionally, where one is screening for bioavailability, e.g.,transport, biological barriers are optionally included. The term“biological barriers” generally refers to cellular or membranous layerswithin biological systems, or synthetic models thereof. Examples of suchbiological barriers include the epithelial and endothelial layers, e.g.vascular endothelia and the like.

[0039] Biological responses are often triggered and/or controlled by thebinding of a receptor to its ligand. For example, interaction of growthfactors, i.e., EGF, FGF, PDGF, etc., with their receptors stimulates awide variety of biological responses including, e.g., cell proliferationand differentiation, activation of mediating enzymes, stimulation ofmessenger turnover, alterations in ion fluxes, activation of enzymes,changes in cell shape and the alteration in genetic expression levels.Accordingly, control of the interaction of the receptor and its ligandmay offer control of the biological responses caused by thatinteraction.

[0040] Accordingly, in one aspect, the present invention will be usefulin screening for compounds that affect an interaction between a receptormolecule and its ligands. As used herein, the term “receptor” generallyrefers to one member of a pair of compounds which specifically recognizeand bind to each other. The other member of the pair is termed a“ligand.” Thus, a receptor/ligand pair may include a typical proteinreceptor, usually membrane associated, and its natural ligand, e.g.,another protein or small molecule. Receptor/ligand pairs may alsoinclude antibody/antigen binding pairs, complementary nucleic acids,nucleic acid associating proteins and their nucleic acid ligands. Alarge number of specifically associating biochemical compounds are wellknown in the art and can be utilized in practicing the presentinvention.

[0041] Traditionally, methods for screening for effectors of areceptor/ligand interaction have involved incubating a receptor/ligandbinding pair in the presence of a test compound. The level of binding ofthe receptor/ligand pair is then compared to negative and/or positivecontrols. Where a decrease in normal binding is seen, the test compoundis determined to be an inhibitor of the receptor/ligand binding. Wherean increase in that binding is seen, the test compound is determined tobe an enhancer or inducer of the interaction.

[0042] In the interest of efficiency, screening assays have typicallybeen set up in multiwell reaction plates, e.g., multi-well microplates,which allow for the simultaneous, parallel screening of large numbers oftest compounds.

[0043] A similar, and perhaps overlapping, set of biochemical systemsincludes the interactions between enzymes and their substrates. The term“enzyme” as used herein, generally refers to a protein which acts as acatalyst to induce a chemical change in other compounds or “substrates.”

[0044] Typically, effectors of an enzyme's activity toward its substrateare screened by contacting the enzyme with a substrate in the presenceand absence of the compound to be screened and under conditions optimalfor detecting changes in the enzyme's activity. After a set time forreaction, the mixture is assayed for the presence of reaction productsor a decrease in the amount of substrate. The amount of substrate thathas been catalyzed is them compared to a control, i.e., enzyme contactedwith substrate in the absence of test compound or presence of a knowneffector. As above, a compound that reduces the enzymes activity towardits substrate is termed an “inhibitor,” whereas a compound thataccentuates that activity is termed an “inducer.”

[0045] Generally, the various screening methods encompassed by thepresent invention involve the serial introduction of a plurality of testcompounds into a microfluidic device. Once injected into the device, thetest compound is screened for effect on a biological system using acontinuous serial or parallel assay orientation.

[0046] As used herein, the term “test compound” refers to the collectionof compounds that are to be screened for their ability to affect aparticular biochemical system. Test compounds may include a wide varietyof different compounds, including chemical compounds, mixtures ofchemical compounds, e.g., polysaccharides, small organic or inorganicmolecules, biological macromolecules, e.g., peptides, proteins, nucleicacids, or an extract made from biological materials such as bacteria,plants, fungi, or animal cells or tissues, naturally occurring orsynthetic compositions. Depending upon the particular embodiment beingpracticed, the test compounds are provided, e.g., injected, free insolution, or are optionally attached to a carrier, or a solid support,e.g., beads. A number of suitable solid supports are employed forimmobilization of the test compounds. Examples of suitable solidsupports include agarose, cellulose, dextran (commercially available as,i.e., Sephadex, Sepharose) carboxymethyl cellulose, polystyrene,polyethylene glycol (PEG), filter paper, nitrocellulose, ion exchangeresins, plastic films, glass beads, polyaminemethylvinylether maleicacid copolymer, amino acid copolymer, ethylene-maleic acid copolymer,nylon, silk, etc. Additionally, for the methods and apparatusesdescribed herein, test compounds are screened individually, or ingroups. Group screening is particularly useful where hit rates foreffective test compounds are expected to be low such that one would notexpect more than one positive result for a given group. Alternatively,such group screening is used where the effects of different testcompounds are differentially detected in a single system, e.g., throughelectrophoretic separation of the effects, or differential labellingwhich enables separate detection.

[0047] Test compounds are commercially available, or derived from any ofa variety of biological sources apparent to one of skill and asdescribed, supra. In one aspect, a tissue homogenate or blood samplefrom a patient is tested in the assay systems of the invention. Forexample, in one aspect, blood is tested for the presence or activity ofa biologically relevant molecule. For example, the presence and activitylevel of an enzyme are detected by supplying and enzyme substrate to thebiological sample and detecting the formation of a product using anassay systems of the invention. Similarly, the presence of infectiouspathogens (viruses, bacteria, fungi, or the like) or cancerous tumorscan be tested by monitoring binding of a labeled ligand to the pathogenor tumor cells, or a component of the pathogen or tumor such as aprotein, cell membrane, cell extract or the like, or alternatively, bymonitoring the presence of an antibody against the pathogen or tumor inthe patient's blood. For example, the binding of an antibody from apatient's blood to a viral protein such as an HIV protein is a commontest for monitoring patient exposure to the virus. Many assays fordetecting pathogen infection are well known, and are adapted to theassay systems of the present invention.

[0048] Biological samples are derived from patients using well knowntechniques such as venipuncture or tissue biopsy. Where the biologicalmaterial is derived from non-human animals, such as commerciallyrelevant livestock, blood and tissue samples are conveniently obtainedfrom livestock processing plants. Similarly, plant material used in theassays of the invention are conveniently derived from agricultural orhorticultural sources. Alternatively, a biological sample can be from acell or blood bank where tissue and/or blood are stored, or from an invitro source such as a culture of cells. Techniques and methods forestablishing a culture of cells for use as a source for biologicalmaterials are well known to those of skill in the art. Freshney Cultureof Animal Cells, a Manual of Basic Technique Third Edition Wiley-Liss,New York (1994) provides a general introduction to cell culture.

[0049] II. Assay Systems

[0050] As described above, the screening methods of the presentinvention are generally carried out in microfluidic devices or“microlaboratory systems,” which allow for integration of the elementsrequired for performing the assay, automation, and minimal environmentaleffects on the assay system, e.g., evaporation, contamination, humanerror, or the like. A number of devices for carrying out the assaymethods of the invention are described in substantial detail below.However, it will be recognized that the specific configuration of thesedevices will generally vary depending upon the type of assay and/orassay orientation desired. For example, in some embodiments, thescreening methods of the invention can be carried out using amicrofluidic device having two intersecting channels. For more complexassays or assay orientations, multichannel/intersection devices areoptionally employed. The small scale, integratability and self-containednature of these devices allows for virtually any assay orientation to berealized within the context of the microlaboratory system.

[0051] A. Electrokinetic Material Transport

[0052] In preferred aspects, the devices, methods and systems describedherein, employ electrokinetic material transport systems, andpreferably, controlled electrokinetic material transport systems. Asused herein, “electrokinetic material transport systems” include systemswhich transport and direct materials within an interconnected channeland/or chamber containing structure, through the application ofelectrical fields to the materials, thereby causing material movementthrough and among the channel and/or chambers, i.e., cations will movetoward the negative electrode, while anions will move toward thepositive electrode.

[0053] Such electrokinetic material transport and direction systemsinclude those systems that rely upon the electrophoretic mobility ofcharged species within the electric field applied to the structure. Suchsystems are more particularly referred to as electrophoretic materialtransport systems. Other electrokinetic material direction and transportsystems rely upon the electroosmotic flow of fluid and material within achannel or chamber structure which results from the application of anelectric field across such structures. In brief, when a fluid is placedinto a channel which has a surface bearing charged functional groups,e.g., hydroxyl groups in etched glass channels or glassmicrocapillaries, those groups can ionize. In the case of hydroxylfunctional groups, this ionization, e.g., at neutral pH, results in therelease of protons from the surface and into the fluid, creating aconcentration of protons at near the fluid/surface interface, or apositively charged sheath surrounding the bulk fluid in the channel.Application of a voltage gradient across the length of the channel, willcause the proton sheath, as well as the fluid it surrounds, to move inthe direction of the voltage drop, i.e., toward the negative electrode.

[0054] “Controlled electrokinetic material transport and direction,” asused herein, refers to electrokinetic systems as described above, whichemploy active control of the voltages applied at multiple, i.e., morethan two, electrodes. Rephrased, such controlled electrokinetic systemsconcomitantly regulate voltage gradients applied across at least twointersecting channels. Controlled electrokinetic material transport isdescribed in Published PCT Application No. WO 96/04547, to Ramsey, whichis incorporated herein by reference in its entirety for all purposes. Inparticular, the preferred microfluidic devices and systems describedherein, include a body structure which includes at least twointersecting channels or fluid conduits, e.g., interconnected, enclosedchambers, which channels include at least three unintersected termini.The intersection of two channels refers to a point at which two or morechannels are in fluid communication with each other, and encompasses “T”intersections, cross intersections, “wagon wheel” intersections ofmultiple channels, or any other channel geometry where two or morechannels are in such fluid communication. An unintersected terminus of achannel is a point at which a channel terminates not as a result of thatchannel's intersection with another channel, e.g., a “T” intersection.In preferred aspects, the devices will include at least threeintersecting channels having at least four unintersected termini. In abasic cross channel structure, where a single horizontal channel isintersected and crossed by a single vertical channel, controlledelectrokinetic material transport operates to controllably directmaterial flow through the intersection, by providing constraining flowsfrom the other channels at the intersection. For example, assuming onewas desirous of transporting a first material through the horizontalchannel, e.g., from left to right, across the intersection with thevertical channel. Simple electrokinetic material flow of this materialacross the intersection could be accomplished by applying a voltagegradient across the length of the horizontal channel, i.e., applying afirst voltage to the left terminus of this channel, and a second, lowervoltage to the right terminus of this channel, or by allowing the rightterminus to float (applying no voltage). However, this type of materialflow through the intersection would result in a substantial amount ofdiffusion at the intersection, resulting from both the natural diffusiveproperties of the material being transported in the medium used, as wellas convective effects at the intersection.

[0055] In controlled electrokinetic material transport, the materialbeing transported across the intersection is constrained by low levelflow from the side channels, e.g., the top and bottom channels. This isaccomplished by applying a slight voltage gradient along the path ofmaterial flow, e.g., from the top or bottom termini of the verticalchannel, toward the right terminus. The result is a “pinching” of thematerial flow at the intersection, which prevents the diffusion of thematerial into the vertical channel. The pinched volume of material atthe intersection may then be injected into the vertical channel byapplying a voltage gradient across the length of the vertical channel,i.e., from the top terminus to the bottom terminus. In order to avoidany bleeding over of material from the horizontal channel during thisinjection, a low level of flow is directed back into the side channels,resulting in a “pull back” of the material from the intersection.

[0056] In addition to pinched injection schemes, controlledelectrokinetic material transport is readily utilized to create virtualvalves which include no mechanical or moving parts. Specifically, withreference to the cross intersection described above, flow of materialfrom one channel segment to another, e.g., the left arm to the right armof the horizontal channel, can be efficiently regulated, stopped andreinitiated, by a controlled flow from the vertical channel, e.g., fromthe bottom arm to the top arm of the vertical channel. Specifically, inthe ‘off’ mode, the material is transported from the left arm, throughthe intersection and into the top arm by applying a voltage gradientacross the left and top termini. A constraining flow is directed fromthe bottom arm to the top arm by applying a similar voltage gradientalong this path (from the bottom terminus to the top terminus). Meteredamounts of material are then dispensed from the left arm into the rightarm of the horizontal channel by switching the applied voltage gradientfrom left to top, to left to right. The amount of time and the voltagegradient applied dictates the amount of material that will be dispensedin this manner. Although described for the purposes of illustration withrespect to a four way, cross intersection, these controlledelectrokinetic material transport systems can be readily adapted formore complex interconnected channel networks, e.g., arrays ofinterconnected parallel channels.

[0057] B. Continuous Flow Assay Systems

[0058] In one preferred aspect, the methods and apparatuses of theinvention are used in screening test compounds using a continuous flowassay system. Generally, the continuous flow assay system can be readilyused in screening for inhibitors or inducers of enzymatic activity, orfor agonists or antagonists of receptor-ligand binding. In brief, thecontinuous flow assay system involves the continuous flow of theparticular biochemical system along a microfabricated channel. As usedherein, the term “continuous” generally refers to an unbroken orcontiguous stream of the particular composition that is beingcontinuously flowed. For example, a continuous flow may include aconstant fluid flow having a set velocity, or alternatively, a fluidflow which includes pauses in the flow rate of the overall system, suchthat the pause does not otherwise interrupt the flow stream. Thefunctioning of the system is indicated by the production of a detectableevent or signal. In one preferred embodiment, such detectable signalsinclude optically detectable chromophoric or fluorescent signals thatare associated with the functioning of the particular model system used.For enzyme systems, such signals will generally be produced by productsof the enzyme's catalytic action, e.g., on a chromogenic or fluorogenicsubstrate. For binding systems, e.g., receptor ligand interactions,signals will typically involve the association of a labeled ligand withthe receptor, or vice versa.

[0059] A wide variety of other detectable signals and labels can also beused in the assays and apparatuses of the invention. In addition to thechromogenic and fluorogenic labels described above, radioactive decay,electron density, changes in pH, solvent viscosity, temperature and saltconcentration are also conveniently measured.

[0060] More generally, labels are commonly detectable by spectroscopic,photochemical, biochemical, immunochemical, or chemical means. Forexample, useful nucleic acid labels include 32P, 35S, fluorescent dyes,electron-dense reagents, enzymes (e.g., as commonly used in an ELISA),biotin, dioxigenin, or haptens and proteins for which antisera ormonoclonal antibodies are available. A wide variety of labels suitablefor labeling biological components are known and are reportedextensively in both the scientific and patent literature, and aregenerally applicable to the present invention for the labeling ofbiological components. Suitable labels include radionucleotides,enzymes, substrates, cofactors, inhibitors, fluorescent moieties,chemiluminescent moieties, magnetic particles, and the like. Labelingagents optionally include e.g., monoclonal antibodies, polyclonalantibodies, proteins, or other polymers such as affinity matrices,carbohydrates or lipids. Detection proceeds by any of a variety of knownmethods, including spectrophotometric or optical tracking of radioactiveor fluorescent markers, or other methods which track a molecule basedupon size, charge or affinity. A detectable moiety can be of anymaterial having a detectable physical or chemical property. Suchdetectable labels have been well-developed in the field of gelelectrophoresis, column chromatograpy, solid substrates, spectroscopictechniques, and the like, and in general, labels useful in such methodscan be applied to the present invention. Thus, a label is anycomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, electrical, optical thermal, or chemical means. Usefullabels in the present invention include fluorescent dyes (e.g.,fluorescein isothiocyanate, Texas red, rhodamine, and the like),radiolabels (e.g., 3H, 125I, 35S, 14C, 32P or 33P), enzymes (e.g., LacZ,CAT, horse radish peroxidase, alkaline phosphatase and others, commonlyused as detectable enzymes, either as marker products or as in anELISA), nucleic acid intercalators (e.g., ethidium bromide) andcolorimetric labels such as colloidal gold or colored glass or plastic(e.g. polystyrene, polypropylene, latex, etc.) beads.

[0061] Fluorescent labels are particularly preferred labels. Preferredlabels are typically characterized by one or more of the following: highsensitivity, high stability, low background, low environmentalsensitivity and high specificity in labeling.

[0062] Fluorescent moieties, which are incorporated into the labels ofthe invention, are generally are known, including 1- and2-aminonaphthalene, p,p′—diaminostilbenes, pyrenes, quaternaryphenanthridine salts, 9-aminoacridines, p,p′-diaminobenzophenone imines,anthracenes, oxacarbocyanine, merocyanine, 3-aminoequilenin, perylene,bis-benzoxazole, bis-p-oxazolyl benzene, 1,2-benzophenazin, retinol,bis-3-aminopyridinium salts, hellebrigenin, tetracycline, sterophenol,benzimidazolylphenylamine, 2-oxo-3-chromen, indole, xanthen,7-hydroxycoumarin, phenoxazine, calicylate, strophanthidin, porphyrins,triarylmethanes and flavin. Individual fluorescent compounds which havefunctionalities for linking to an element desirably detected in anapparatus or assay of the invention, or which can be modified toincorporate such functionalities include, e.g., dansyl chloride;fluoresceins such as 3,6-dihydroxy-9-phenylxanthhydrol;rhodamineisothiocyanate; N-phenyl 1-amino-8—sulfonatonaphthalene;N-phenyl 2-amino-6-sulfonatonaphthalene;4-acetamido-4-isothiocyanato-stilbene-2,2′-disulfonic acid;pyrene-3-sulfonic acid; 2-toluidinonaphthalene-6-sulfonate;N-phenyl-N-methyl-2-aminoaphthalene-6-sulfonate; ethidium bromide;stebrine; auromine-0,2-(9′-anthroyl)palmitate; dansylphosphatidylethanolamine; N,N′-dioctadecyl oxacarbocyanine: N,N′-dihexyloxacarbocyanine; merocyanine, 4-(3′pyrenyl)stearate;d-3-aminodesoxy-equilenin; 12-(9′-anthroyl)stearate; 2-methylanthracene;9-vinylanthracene; 2,2′(vinylene-p-phenylene)bisbenzoxazole;p-bis(2-(4-methyl-5-phenyl-oxazolyl))benzene;6-dimethylamino-1,2-benzophenazin; retinol; bis(3′-aminopyridinium)1,10-decandiyl diiodide; sulfonaphthylhydrazone of hellibrienin;chlorotetracycline;N-(7-dimethylamino-4-methyl-2-oxo-3-chromenyl)maleimide;N-(p-(2-benzimidazolyl)-phenyl)maleimide; N-(4-fluoranthyl)maleimide;bis(homovanillic acid); resazarin;4-chloro-7-nitro-2,1,3-benzooxadiazole; merocyanine 540; resorufin; rosebengal; and 2,4-diphenyl-3(2H)-furanone. Many fluorescent tags arecommercially available from SIGMA chemical company (Saint Louis, Mo.),Molecular Probes, R&D systems (Minneapolis, Minn.), Pharmacia LKBBiotechnology (Piscataway, N.J.), CLONTECH Laboratories, Inc. (PaloAlto, Calif.), Chem Genes Corp., Aldrich Chemical Company (Milwaukee,Wis.), Glen Research, Inc., GIBCO BRL Life Technologies, Inc.(Gaithersberg, Md.), Fluka Chemica-Biochemika Analytika (Fluka ChemieAG, Buchs, Switzerland), and Applied Biosystems (Foster City, Calif.) aswell as other commercial sources known to one of skill.

[0063] Desirably, fluorescent labels absorb light above about 300 nm,preferably about 350 nm, and more preferably above about 400 nm, usuallyemitting at wavelengths greater than about 10 nm higher than thewavelength of the light absorbed. It should be noted that the absorptionand emission characteristics of the bound label may differ from theunbound label. Therefore, when referring to the various wavelengthranges and characteristics of the labels, it is intended to indicate thelabels as employed and not the label which is unconjugated andcharacterized in an arbitrary solvent.

[0064] Fluorescent labels are one preferred class of detectable labels,in part because by irradiating a fluorescent label with light, one canobtain a plurality of emissions. Thus, a single label can provide for aplurality of measurable events. Detectable signal may also be providedby chemiluminescent and bioluminescent sources. Chemiluminescent sourcesinclude a compound which becomes electronically excited by a chemicalreaction and may then emit light which serves as the detectible signalor donates energy to a fluorescent acceptor. A diverse number offamilies of compounds have been found to provide chemiluminescence undera variety or conditions. One family of compounds is2,3-dihydro-1,4—phthalazinedione. The most popular compound is luminol,which is a 5-amino compound. Other members of the family include the5-amino-6,7,8-trirnethoxy- and the dimethylamino[ca]benz analog. Thesecompounds can be made to luminesce with alkaline hydrogen peroxide orcalcium hypochlorite and base. Another family of compounds is the2,4,5-triphenylimidazoles, with lophine as the common name for theparent product. Chemiluminescent analogs include para-dimethylamino and-methoxy substituents. Chemiluminescence may also be obtained withoxalates, usually oxalyl active esters, e.g., p-nitrophenyl and aperoxide, e.g., hydrogen peroxide, under basic conditions. Other usefulchemiluminescent compounds are also known and available, including-N-alkyl acridinum esters (basic H₂O₂) and dioxetanes. Alternatively,luciferins may be used in conjunction with luciferase or lucigenins toprovide bioluminescence.

[0065] The label is coupled directly or indirectly to a molecule to bedetected (a product, substrate, enzyme, or the like) according tomethods well known in the art. As indicated above, a wide variety oflabels are used, with the choice of label depending on the sensitivityrequired, ease of conjugation of the compound, stability requirements,available instrumentation, and disposal provisions. Non radioactivelabels are often attached by indirect means. Generally, a ligandmolecule (e.g., biotin) is covalently bound to a polymer. The ligandthen binds to an anti-ligand (e.g., streptavidin) molecule which iseither inherently detectable or covalently bound to a signal system,such as a detectable enzyme, a fluorescent compound, or achemiluminescent compound. A number of ligands and anti-ligands can beused. Where a ligand has a natural anti-ligand, for example, biotin,thyroxine, and cortisol, it can be, used in conjunction with labeled,anti-ligands. Alternatively, any haptenic or antigenic compound can beused in combination with an antibody. Labels can also be conjugateddirectly to signal generating compounds, e.g., by conjugation with anenzyme or fluorophore. Enzymes of interest as labels will primarily behydrolases, particularly phosphatases, esterases and glycosidases, oroxidoreductases, particularly peroxidases. Fluorescent compounds includefluorescein and its derivatives, rhodamine and its derivatives, dansyl,umbelliferone, etc. Chemiluminescent compounds include luciferin, and2,3-dihydrophthalazinediones, e.g., luminol. Means of detecting labelsare well known to those of skill in the art. Thus, for example, wherethe label is a radioactive label, means for detection include ascintillation counter or photographic film as in autoradiography. Wherethe label is a fluorescent label, it may be detected by exciting thefluorochrome with the appropriate wavelength of light and detecting theresulting fluorescence, e.g., by microscopy, visual inspection, viaphotographic film, by the use of electronic detectors such as digitalcameras, charge coupled devices (CCDs) or photomultipliers andphototubes, and the like. Fluorescent labels and detection techniques,particularly microscopy and spectroscopy are preferred. Similarly,enzymatic labels are detected by providing appropriate substrates forthe enzyme and detecting the resulting reaction product. Finally, simplecolorimetric labels are often detected simply by observing the colorassociated with the label. For example, conjugated gold often appearspink, while various conjugated beads appear the color of the bead.

[0066] In preferred aspects, the continuous system generates a constantsignal which varies only when a test compound is introduced that affectsthe system. Specifically, as the system components flow along thechannel, they will produce a relatively constant signal level at adetection zone or window of the channel. Test compounds are periodicallyintroduced into the channel and mixed with the system components. Wherethose test compounds have an effect on the system, it will cause adeviation from the constant signal level at the detection window. Thisdeviation may then be correlated to the particular test compoundscreened.

[0067] One embodiment of a device for use in a serial or continuousassay geometry is shown in FIG. 1. As shown, the overall device 100 isfabricated in a planar substrate 102. Suitable substrate materials aregenerally selected based upon their compatibility with the conditionspresent in the particular operation to be performed by the device. Suchconditions can include extremes of pH, temperature, salt concentration,and application of electrical fields. Additionally, substrate materialsare also selected for their inertness to critical components of ananalysis or synthesis to be carried out by the device.

[0068] Examples of useful substrate materials include, e.g., glass,quartz and silicon as well as polymeric substrates, e.g. plastics. Inthe case of conductive or semi-conductive substrates, it will generallybe desirable to include an insulating layer on the substrate. This isparticularly important where the device incorporates electricalelements, e.g., electrical material and fluid direction systems, sensorsand the like. In the case of polymeric substrates, the substratematerials are optionally rigid, semi-rigid, or non-rigid, opaque,semi-opaque or transparent, depending upon the use for which they areintended. For example, devices which include an optical or visualdetection element, will generally be fabricated, at least in part, fromtransparent materials to allow, or at least, facilitate that detection.Alternatively, transparent windows of, e.g., glass or quartz, areoptionally incorporated into the device for these types detectionelements. Additionally, the polymeric materials may have linear orbranched backbones, and are optionally crosslinked or non-crosslinked.Examples of particularly preferred polymeric materials include, e.g.,polydimethylsiloxanes (PDMS), polyurethane, polyvinylchloride (PVC)polystyrene, polysulfone, polycarbonate and the like.

[0069] The device shown in FIG. 1 includes a series of channels 110,112, and optional reagent channel 114, fabricated into the surface ofthe substrate. At least one of these channels will typically have verysmall cross sectional dimensions, e.g., in the range of from about 0.1μm to about 500 μm. Preferably the cross-sectional dimensions of thechannels will be in the range of from about 0.1 to about 200 μm and morepreferably in the range of from about 0.1 to about 100 μm. Inparticularly preferred aspects, each of the channels will have at leastone cross-sectional dimension in the range of from about 0.1 μm to about100 μm. Although generally shown as straight channels, it will beappreciated that in order to maximize the use of space on a substrate,serpentine, saw tooth or other channel geometries, to incorporateeffectively longer channels in shorter distances.

[0070] Manufacturing of these microscale elements into the surface ofthe substrates may generally be carried out by any number ofmicrofabrication techniques that are well known in the art. For example,lithographic techniques are optionally employed in fabricating, e.g.,glass, quartz or silicon substrates, using methods well known in thesemiconductor manufacturing industries such as photolithographicetching, plasma etching or wet chemical etching. Alternatively,micromachining methods such as laser drilling, micromilling and the likeare optionally employed. Similarly, for polymeric substrates, well knownmanufacturing techniques may also be used. These techniques includeinjection molding or stamp molding methods where large numbers ofsubstrates are optionally produced using, e.g., rolling stamps toproduce large sheets of microscale substrates or polymer microcastingtechniques where the substrate is polymerized within a micromachinedmold.

[0071] The devices will typically include an additional planar elementwhich overlays the channeled substrate enclosing and fluidly sealing thevarious channels to form conduits. Attaching the planar cover element isachieved by a variety of means, including, e.g., thermal bonding,adhesives or, in the case of certain substrates, e.g., glass, orsemi-rigid and non-rigid polymeric substrates, a natural adhesionbetween the two components. The planar cover element may additionally beprovided with access ports and/or reservoirs for introducing the variousfluid elements needed for a particular screen.

[0072] The device shown in FIG. 1 also includes reservoirs 104, 106 and108, disposed and fluidly connected at the ends of the channels 110 and114. As shown, sample channel 112, is used to introduce the plurality ofdifferent test compounds into the device. As such, this channel willgenerally be fluidly connected to a source of large numbers of separatetest compounds that will be individually introduced into the samplechannel 112 and subsequently into channel 110.

[0073] The introduction of large numbers of individual, discrete volumesof test compounds into the sample is carried out by a number of methods.For example, micropipettors are optionally used to introduce the testcompounds into the device. In preferred aspects, an electropipettor isused which is fluidly connected to sample channel 112. An example ofsuch an electropipettor is described in, e.g., U.S. patent applicationSer. No. 08/671,986, filed Jun. 28, 1996 (Attorney Docket No.017646-000500) the disclosure of which is hereby incorporated herein byreference in its entirety for all purposes. Generally, thiselectropipettor utilizes electroosmotic fluid direction as describedherein, to alternately sample a number of test compounds, or “subjectmaterials,” and spacer compounds. The pipettor then delivers individual,physically isolated sample or test compound volumes in subject materialregions, in series, into the sample channel for subsequent manipulationwithin the device. Individual samples are typically separated by aspacer region of low ionic strength spacer fluid. These low ionicstrength spacer regions have higher voltage drop over their length thando the higher ionic strength subject material or test compound regions,thereby driving the electrokinetic pumping. On either side of the testcompound or subject material region, which is typically in higher ionicstrength solution, are fluid regions referred to as first spacer regions(also referred to as “guard bands”), that contact the interface of thesubject material regions. These first spacer regions typically comprisea high ionic strength solution to prevent migration of the sampleelements into the lower ionic strength fluid regions, or second spacerregion, which would result in electrophoretic bias. The use of suchfirst and second spacer regions is described in greater detail in U.S.patent application Ser. No. 08/671,986, filed Jun. 28, 1996, (AttorneyDocket No. 017646-000500) which is incorporated herein by reference.

[0074] Alternatively, the sample channel 112 is optionally individuallyfluidly connected to a plurality of separate reservoirs via separatechannels. The separate reservoirs each contain a separate test compoundwith additional reservoirs being provided for appropriate spacercompounds. The test compounds and/or spacer compounds are thentransported from the various reservoirs into the sample channels usingappropriate material direction schemes. In either case, it generally isdesirable to separate the discrete sample volumes, or test compounds,with appropriate spacer regions.

[0075] As shown, the device also includes a detection window or zone 116at which a signal from the biochemical system is optionally monitored.This detection window typically will include a transparent coverallowing visual or optical observation and detection of the assayresults, e.g., observation of a colorometric or fluorometric response.

[0076] In particularly preferred aspects, monitoring of the signals atthe detection window is achieved using an optical detection system. Forexample, fluorescence based signals are typically monitored using, e.g.,laser activated fluorescence detection systems which employ a laserlight source at an appropriate wavelength for activating the fluorescentindicator within the system. Fluorescence is then detected using anappropriate detector element, e.g., a photomultiplier tube (PMT).Similarly, for screens employing colorometric signals,spectrophotometric detection systems which direct a light source at thesample are optionally used, providing a measurement of absorbance ortransmissivity of the sample.

[0077] In alternative aspects, the detection system may comprisenon-optical detectors or sensors for detecting a particularcharacteristic of the system disposed within detection window 116. Suchsensors may include temperature, conductivity, potentiometric (pH,ions), amperometric (for compounds that are oxidized or reduced, e.g.,O₂, H₂O₂ , I ₂, oxidizable/reducible organic compounds, and the like).

[0078] In operation, a flowable first component of a biological system,e.g., a fluid comprising a receptor or enzyme, is placed in reservoir104. This first component is flowed through main channel 110, past thedetection window, 116, and toward waste reservoir 108. A secondcomponent of the biochemical system, e.g., a ligand or substrate, isconcurrently flowed into the main channel 110 from the side channel 114,whereupon the first and second components mix and are able to interact.Deposition of these elements within the device is carried out in anumber of ways. For example, the enzyme and substrate, or receptor andligand solutions can be introduced into the device through open orsealable access ports in the planar cover. Alternatively, thesecomponents are optionally added to their respective reservoirs duringmanufacture of the device. In the case of such pre-added components, itis desirable to provide these components in a stabilized form to allowfor prolonged shelf-life of the device. For example, theenzyme/substrate or receptor/ligand components are optionally providedwithin the device in lyophilized form. Prior to use, these componentsare easily reconstituted by introducing a buffer solution into thereservoirs. Alternatively, the components are lyophilized withappropriate buffering salts, whereby simple water addition is all thatis required for reconstitution.

[0079] As noted above, the interaction of the first and secondcomponents is typically accompanied by a detectable signal. For example,in those embodiments where the first component is an enzyme and thesecond a substrate, the substrate is a chromogenic or fluorogenicsubstrate which produces an optically detectable signal when the enzymeacts upon the substrate. In the case where the first component is areceptor and the second is a ligand, either the ligand or the receptoroptionally includes a detectable signal. In either event, the mixtureand flow rate of compounds will typically remain constant such that theflow of the mixture of the first and second components past thedetection window 116 will produce a steady-state signal. By “steadystate signal” is generally meant a signal that has a regular,predictable signal intensity profile. As such, the steady-state signalmay include signals having a constant signal intensity, oralternatively, a signal with a regular periodic intensity, against whichvariations in the normal signal profile is measured. This latter signalis generated in cases where fluid flow is periodically interrupted for,e.g., loading additional test compounds, as described in the descriptionof the continuous flow systems. Although the signal produced in theabove-described enzymatic system will vary along the length of thechannel, i.e., increasing with time of exposure as the enzyme convertsthe fluorogenic substrate to the fluorescent product, the signal at anyspecific point along the channel will remain constant, given a constantflow rate.

[0080] From sample channel 112, test compounds is periodically orserially introduced into the main channel 110 and into the stream offirst and second components as fluid regions containing the testcompound, also referred to as the “subject material regions.” Wherethese test compounds have an effect on the interaction of the first andsecond elements, it will produce a deviation in the signal detected atthe detection window corresponding to the subject material region. Asnoted above, typically, the various different test compounds to beinjected through channel 112 will be separated by a first and evensecond spacer fluid regions to allow differentiation of the effects, orlack of effects, from one test compound to another. In those embodimentswhere electroosmotic fluid direction systems are employed, the spacerfluid regions may also function to reduce any electrophoretic bias thatcan occur within the test sample. The use of these spacer regions todrive the electroosmotic flow of fluids, as well as in the generalelimination of electrophoretic bias within the sample or test compoundor subject material regions is substantially described in U.S. patentapplication Ser. No. 08/671,986, filed Jun. 28, 1996 (Attorney DocketNo. 017646-000500), previously incorporated herein by reference.

[0081] By way of example, a steady, continuous flow of enzyme andfluorogenic substrate through main channel 110 will produce a constantfluorescent signal at the detection window 116. Where a test compoundinhibits the enzyme, introduction of a test compound, i.e., in a subjectmaterial region, will produce a momentary but detectable drop in thelevel of signal at the detection window corresponding with that subjectmaterial region. The timing of the drop in signal can then be correlatedwith a particular test compound based upon a known injection todetection time-frame. Specifically, the time required for an injectedcompound to produce an observed effect can be readily determined usingpositive controls.

[0082] For receptor/ligand systems, a similar variation in the steadystate signal may also be observed. Specifically, the receptor and itsfluorescent ligand can be made to have different flow rates along thechannel. This can be accomplished by incorporating size exclusionmatrices within the channel, or, in the case of electroosmotic methods,altering the relative electrophoretic mobility of the two compounds sothat the receptor flows more rapidly down the channel. Again, this isaccomplished through the use of size exclusion matrices, or through theuse of different surface charges in the channel which will result indifferential flow rates of charge-varied compounds. Where a testcompound binds to the receptor, it will result in a dark pulse in thefluorescent signal followed by a brighter pulse. Without being bound toa particular theory of operation, it is believed that the steady statesignal is a result of both free fluorescent ligand, and fluorescentligand bound to the receptor. The bound ligand is traveling at the sameflow rate as the receptor while the unbound ligand is traveling moreslowly. Where the test compound inhibits the receptor-ligandinteraction, the receptor will not ‘bring along’ the fluorescent ligand,thereby diluting the fluorescent ligand in the direction of flow, andleaving an excess of free fluorescent ligand behind. This results in atemporary reduction in the steady-state signal, followed by a temporaryincrease in fluorescence. Alternatively, schemes similar to thoseemployed for the enzymatic system is employed, where there is a signalthat reflects the interaction of the receptor with its ligand. Forexample, pH indicators which indicate pH effects of receptor-ligandbinding is incorporated into the device along with the biochemicalsystem, i.e., in the form of encapsulated cells, whereby slight pHchanges resulting from binding can be detected. See Weaver, et al.,Bio/Technology (1988) 6:1084-1089. Additionally, one can monitoractivation of enzymes resulting from receptor ligand binding, e.g.,activation of kinases, or detect conformational changes in such enzymesupon activation, e.g., through incorporation of a fluorophore which isactivated or quenched by the conformational change to the enzyme uponactivation.

[0083] Flowing and direction of fluids within the microscale fluidicdevices is carried out by a variety of methods. For example, the devicesmay include integrated microfluidic structures, such as micropumps andmicrovalves, or external elements, e.g., pumps and switching valves, forthe pumping and direction of the various fluids through the device.Examples of microfluidic structures are described in, e.g., U.S. Pat.Nos. 5,271,724, 5,277,556, 5,171,132, and 5,375,979. See also, PublishedU.K. Patent Application No. 2 248 891 and Published European PatentApplication No. 568 902.

[0084] Although microfabricated fluid pumping and valving systems arereadily employed in the devices of the invention, the cost andcomplexity associated with their manufacture and operation can generallyprohibit their use in mass-produced disposable devices as are envisionedby the present invention. For that reason, in particularly preferredaspects, the devices of the invention will typically include anelectroosmotic fluid direction system. Such fluid direction systemscombine the elegance of a fluid direction system devoid of moving parts,with an ease of manufacturing, fluid control and disposability. Examplesof particularly preferred electroosmotic fluid direction systemsinclude, e.g., those described in International Patent Application No.WO 96/04547 to Ramsey et al., which is incorporated herein by referencein its entirety for all purposes.

[0085] In brief, these fluidic control systems typically includeelectrodes disposed within the reservoirs that are placed in fluidconnection with the plurality of intersecting channels fabricated intothe surface of the substrate. The materials stored in the reservoirs aretransported through the channel system delivering appropriate volumes ofthe various materials to one or more regions on the substrate in orderto carry out a desired screening assay.

[0086] Fluid and materials transport and direction is accomplishedthrough electroosmosis or electrokinesis. In brief, when an appropriatematerial, typically comprising a fluid, is placed in a channel or otherfluid conduit having functional groups present at the surface, thosegroups can ionize. For example, where the surface of the channelincludes hydroxyl functional groups at the surface, protons can leavethe surface of the channel and enter the fluid. Under such conditions,the surface will possess a net negative charge, whereas the fluid willpossess an excess of protons or positive charge, particularly localizednear the interface between the channel surface and the fluid. Byapplying an electric field along the length of the channel, cations willflow toward the negative electrode. Movement of the positively chargedspecies in the fluid pulls the solvent with them. The steady statevelocity of this fluid movement is generally given by the equation:$v = \frac{\varepsilon \quad \xi \quad E}{4{\pi\eta}}$

[0087] where v is the solvent velocity, ε is the dielectric constant ofthe fluid, ξ is the zeta potential of the surface, E is the electricfield strength, and η is the solvent viscosity. Thus, as can be easilyseen from this equation, the solvent velocity is directly proportionalto the surface potential.

[0088] To provide appropriate electric fields, the system generallyincludes a voltage controller that is capable of applying selectablevoltage levels, simultaneously, to each of the reservoirs, includingground. Such a voltage controller can be implemented using multiplevoltage dividers and multiple relays to obtain the selectable voltagelevels. Alternatively, multiple, independent voltage sources areoptionally used. The voltage controller is electrically connected toeach of the reservoirs via an electrode positioned or fabricated withineach of the plurality of reservoirs.

[0089] Incorporating this electroosmotic fluid direction system into thedevice shown in FIG. 1 involves incorporation of an electrode withineach of the reservoirs 104, 106 and 108, and at the terminus of samplechannel 112 or at the terminus of any fluid channels connected thereto,whereby the electrode is in electrical contact with the fluid disposedin the respective reservoir or channel. Substrate materials are alsoselected to produce channels having a desired surface charge. In thecase of glass substrates, the etched channels will possess a netnegative charge resulting from the ionized hydroxyls naturally presentat the surface. Alternatively, surface modifications are optionallyemployed to provide an appropriate surface charge, e.g., coatings,derivatization, e.g., silanation, or impregnation of the surface toprovide appropriately charged groups on the surface. Examples of suchtreatments are described in, e.g., Provisional Patent Application SerialNo. 60/015,498, filed Apr. 16, 1996 (Attorney Docket No. 017646-002600)which is hereby incorporated herein by reference in its entirety for allpurposes.

[0090] In brief, suitable substrate materials are generally selectedbased upon their compatibility with the conditions present in theparticular operation to be performed by the device. Such conditions caninclude extremes of pH, temperature and salt concentration.Additionally, substrate materials are also selected for their inertnessto critical components of an analysis or synthesis to be carried out bythe device. Polymeric substrate materials may be rigid, semi-rigid, ornon-rigid, opaque, semi-opaque or transparent, depending upon the usefor which they are intended. For example, devices which include anoptical or visual detection element, will generally be fabricated, atleast in part, from a transparent polymeric material to facilitate thatdetection. Alternatively, transparent windows of, e.g. glass or quartz,may be incorporated into the device for these detection elements.Additionally, the polymeric materials may have linear or branchedbackbones, and may be crosslinked or non-crosslinked. Examples ofpolymeric materials include, e.g., Acrylics, especially PMMAs(polymethylmethacrylates); exemplar acrylics include e.g., Acrylite M-30or Acrylite L-40 available from CYRO Industries, Rockaway, N.J., orPLEXIGLAS VS UVT available from Autohaas North America; polycarbonates(e.g., Makrolon CD-2005 available from The Plastics and Rubber divisionof Mobay Corporation (Pittsburg, Pa.) or Bayer Corporation, or LEXAN OQ1020L or LEXAN OQ 1020, both available from GE Plastics)polydimethylsiloxanes (PDMS), polyurethane, polyvinylchloride (PVC)polystyrene, polysulfone, polycarbonate and the like. Optical,mechanical, thermal, electrical, and chemical resistance properties formany plastics are well known (and are generally available from themanufacturer), or can easily be determined by standard assays.

[0091] As described herein, the electrokinetic fluid control systemsemployed in the devices of the present invention generally utilize asubstrate having charged functional groups at its surface, such as thehydroxyl groups present on glass surfaces. As described, devices of thepresent invention can also employ plastic or other polymeric substrates.In general, these substrate materials have hydrophobic surfaces. As aresult, use of electrokinetic fluid control systems in devices utilizingpolymeric substrates used in the present invention typically employsmodification of the surfaces of the substrate that are in contact withfluids.

[0092] Surface modification of polymeric substrates may take on avariety of different forms. For example, surfaces may be coated with anappropriately charged material. For example, surfactants with chargedgroups and hydrophobic tails are desirable coating materials. In short,the hydrophobic tails will localize to the hydrophobic surface of thesubstrate, thereby presenting the charged head group at the fluid layer.

[0093] In one embodiment, preparation of a charged surface on thesubstrate involves the exposure of the surface to be modified, e.g., thechannels and/or reaction chambers, to an appropriate solvent whichpartially dissolves or softens the surface of the polymeric substrate. Adetergent is then contacted with the partially dissolved surface. Thehydrophobic portion of the detergent molecules will associate with thepartially dissolve polymer. The solvent is then washed from the surface,e.g., using water, whereupon the polymer surface hardens with thedetergent embedded into the surface, presenting the charged head groupto the fluid interface.

[0094] In alternative aspects, polymeric materials, such aspolydimethylsiloxane, may be modified by plasma irradiation. Inparticular, plasma irradiation of PDMS oxidizes the methyl groups,liberating the carbons and leaving hydroxyl groups in their place,effectively creating a glass-like surface on the polymeric material,with its associated hydroxyl functional groups.

[0095] The polymeric substrate may be rigid, semi-rigid, nonrigid or acombination of rigid and nonrigid elements, depending upon theparticular application for which the device is to be used. In oneembodiment, a substrate is made up of at least one softer, flexiblesubstrate element and at least one harder, more rigid substrate element,one of which includes the channels and chambers manufactured into itssurface. Upon mating the two substrates, the inclusion of the softelement allows formation of an effective fluid seal for the channels andchambers, obviating the need and problems associated with gluing ormelting more rigid plastic components together.

[0096] A number of additional elements are added to the polymericsubstrate to provide for the electrokinetic fluid control systems. Theseelements may be added either during the substrate formation process,i.e., during the molding or stamping steps, or they may be added duringa separate, subsequent step. These elements typically include electrodesfor the application of voltages to the various fluid reservoirs, and insome embodiments, voltage sensors at the various channel intersectionsto monitor the voltage applied.

[0097] Electrodes may be incorporated as a portion of the moldingprocess. In particular, the electrodes may be patterned within the moldso that upon introduction of the polymeric material into the mold, theelectrodes will be appropriately placed. Alternatively, the electrodesand other elements may be added after the substrate is formed, usingwell known microfabrication methods, e.g., sputtering or controlledvapor deposition methods followed by chemical etching.

[0098] Whether polymeric or other substrates are used, modulatingvoltages are concomitantly applied to the various reservoirs to affect adesired fluid flow characteristic, e.g., continuous flow ofreceptor/enzyme, ligand/substrate toward the waste reservoir with theperiodic introduction of test compounds. Particularly, modulation of thevoltages applied at the various reservoirs can move and direct fluidflow through the interconnected channel structure of the device in acontrolled manner to effect the fluid flow for the desired screeningassay and apparatus.

[0099]FIG. 2A shows a schematic illustration of fluid direction during atypical assay screen. Specifically, shown is the injection of a testcompound (in a subject material region) into a continuous stream of anenzyme-fluorogenic substrate mixture. As shown in FIG. 2A, and withreference to FIG. 1, a continuous stream of enzyme is flowed fromreservoir 104, along main channel 110. Test compounds 120, separated byappropriate spacer regions 121, e.g., low ionic strength spacer regions,are introduced from sample channel 112 into main channel 110. Onceintroduced into the main channel, the test compounds will interact withthe flowing enzyme stream. The mixed enzyme/test compound regions arethen flowed along main channel 110 past the intersection with channel114. A continuous stream of fluorogenic or chromogenic substrate whichis contained in reservoir 106, is introduced into sample channel 110,whereupon it contacts and mixes with the continuous stream of enzyme,including the subject material regions which include the test compounds122. Action of the enzyme upon the substrate will produce an increasinglevel of the fluorescent or chromatic signal. This increasing signal isindicated by the increasing shading within the main channel as itapproaches the detection window. This signal trend will also occurwithin those test compound or subject material regions which have noeffect on the enzyme/substrate interaction, e.g., test compound 126.Where a test compound does have an effect on the interaction of theenzyme and the substrate, a variation will appear in the signalproduced. For example, assuming a fluorogenic substrate, a test compoundwhich inhibits the interaction of the enzyme with its substrate willresult in less fluorescent product being produced within that subjectmaterial region. This will result in a non-fluorescent, or detectablyless fluorescent region within the flowing stream as it passes detectionwindow 116, which corresponds to the subject material region. Forexample, as shown, a subject material region including a test compound128, which is a putative inhibitor of the enzyme-substrate interaction,shows detectably lower fluorescence than the surrounding stream. This isindicated by a lack of shading of subject material region 128.

[0100] A detector adjacent to the detection window monitors the level offluorescent signal being produced by the enzyme's activity on thefluorogenic or chromogenic substrate. This signal remains at arelatively constant level for those test compounds which have no effecton the enzyme-substrate interaction. When an inhibitory compound isscreened, however, it will produce a momentary drop in the fluorescentsignal representing the reduced or inhibited enzyme activity toward thesubstrate. Conversely, inducer compounds, upon screening, produce amomentary increase in the fluorescent signal, corresponding to theincreased enzyme activity toward the substrate.

[0101]FIG. 2B provides a similar schematic illustration of a screen foreffectors of a receptor-ligand interaction. As in FIG. 2A, a continuousstream of receptor is flowed from reservoir 104 through main channel110. Test compounds or subject material regions 150 separated byappropriate spacer fluid regions 121 are introduced into the mainchannel 110 from sample channel 112, and a continuous stream offluorescent ligand from reservoir 106 is introduced from side channel114. Fluorescence is indicated by shading within the channel. As in FIG.2A, the continuous stream of fluorescent ligand and receptor past thedetection window 116 will provide a constant signal intensity. Thesubject material regions in the stream, containing the test compoundswhich have no effect on the receptor-ligand interaction, will providethe same or similar level of fluorescence as the rest of the surroundingstream, e.g., test compound or subject material region 152. However, thepresence of test compounds which possess antagonistic or inhibitoryactivity toward the receptor-ligand interaction will result in lowerlevels of that interaction in those portions of the stream where thosecompounds are located, e.g., test compound or subject material region154. Further, differential flow rates for the receptor bound fluorescentligand and free fluorescent ligand will result in a detectable drop inthe level of fluorescence which corresponds to the dilution of thefluorescence resulting from unbound, faster moving receptor. The drop influorescence is then followed by an increase in fluorescence 156 whichcorresponds to an accumulation of the slower moving, unbound fluorescentligand.

[0102] In some embodiments, it is desirable to provide an additionalchannel for shunting off or extracting the subject material regionreaction mixture from the running buffer and/or spacer regions. This maybe the case where one wishes to keep the reaction elements containedwithin the a discrete fluid region during the reaction, while allowingthese elements to be separated during a data acquisition stage. Asdescribed previously, one can keep the various elements of the reactiontogether in the subject material region that is moving through thereaction channel by incorporating appropriate spacer fluid regionsbetween samples. Such spacer fluid regions are generally selected toretain the samples within their original subject material regions, i.e.,not allowing smearing of the sample into the spacer regions, even duringprolonged reaction periods. However, this goal can be at odds with thoseassays which are based upon the separation of elements of the assay,e.g., ligand-receptor assays described above, or where a reactionproduct must be separated in a capillary. Thus, it may be desirable toremove those elements which prevented such separation during the initialportions of the fluid direction.

[0103] A schematic illustration of one embodiment of a device 500 forperforming this sample or subject material shunting or extraction isshown in FIG. 5. As shown, the subject materials or test compounds 504are introduced to the device or chip via the sample channel 512. Again,these are typically introduced via an appropriate injection device 506,e.g., a capillary pipettor. The ionic strength and lengths of the firstspacer regions 508 and second spacer regions 502 are selected such thatthose samples with the highest electrophoretic mobility will not migratethrough the first spacer regions 508 into the second spacer regions 502in the length of time that it takes the sample to travel down thereaction channel.

[0104] Assuming a receptor ligand assay system, test compounds pass intothe device 500 and into reaction channel 510, where they are firstcombined with the receptor. The test compound/receptor, in the form ofthe subject material regions, are flowed along the reaction channel inthe incubation zone 510 a. Following this initial incubation, the testcompound/receptor mix is combined with a labelled ligand (e.g.,fluorescent ligand) whereupon this mixture flows along the secondincubation region 510 b of reaction channel 510. The lengths of theincubation regions and the flow rates of the system (determined by thepotentials applied at each of the reservoirs 514, 516, 518, 520, 522,and at the terminus of sample channel 512) determine the time ofincubation of the receptor with the fluorescent ligand and testcompound. The ionic strengths of the solutions containing the receptorsand fluorescent ligands, as well as the flow rates of material from thereservoirs housing these elements into the sample channel are selectedso as to not interfere with the first and second spacer regions.

[0105] The isolated subject material regions containing receptor,fluorescent ligand and test compound are flowed along the reactionchannel 510 by the application of potentials at, e.g., reservoirs 514,516, 518 and at the terminus of sample channel 512. Potentials are alsoapplied at reservoirs 520 and 522, at the opposite ends of separationchannel 524, to match the potentials at the two ends of the transferchannel, so that the net flow across the transfer channel is zero. Asthe subject material region passes the intersection of reaction channel510 and transfer channel 526, the potentials are allowed to float atreservoirs 518 and 522, whereupon the potentials applied at reservoirs514, 516, 520, and at the terminus of sample channel 512, result in thesubject material region being shunted through transfer channel 526 andinto separation channel 524. Once in the separation channel, theoriginal potentials are reapplied to all of the reservoirs to stop thenet fluid flow through transfer channel 526. The diversion of thesubject material can then be repeated with each subsequent subjectmaterial region. Within the separation channel, the subject materialregion is exposed to different conditions than those of the reactionchannel. For example, a different flow rate may be used, capillarytreatments may allow for separation of differentially charged ordifferent sized species, and the like. In a preferred aspect, thesubject material is shunted into the separation channel to place thesubject material into a capillary filled with high ionic strengthbuffer, i.e., to remove the low ionic strength spacer regions, therebyallowing separation of the various sample components outside theconfines of the original subject material region. For example, in thecase of the above-described receptor/ligand screen, the receptor/ligandcomplex may have a different electrophoretic mobility from the ligandalone, in the transfer channel, thereby allowing more pronouncedseparation of the complex from the ligand, and its subsequent detection.

[0106] This device may be particularly useful for screening testcompounds serially injected into the device, but employing a parallelassay geometry, once the samples are introduced into the device, toallow for increased throughput.

[0107] In operation, test compounds in discrete subject materialregions, are serially introduced into the device, separated as describedabove, and flowed along the transverse sample injection channel 304until the separate subject material regions are adjacent theintersection of the sample channel 304 with the parallel reactionchannels 310-324. As shown in FIGS. 4A-4F, the test compounds areoptionally provided immobilized on individual beads. In those caseswhere the test compounds are immobilized on beads, the parallel channelsare optionally fabricated to include bead resting wells 326-338 at theintersection of the reaction channels with the sample injection channel304. Arrows 340 indicate the net fluid flow during this type ofsample/bead injection. As individual beads settle into a resting well,fluid flow through that particular channel will be generally restricted.The next bead in the series following the unrestricted fluid flow, thenflows to the next available resting well to settle in place.

[0108] Once in position adjacent to the intersection of the parallelreaction channel and the sample injection channel, the test compound isdirected into its respective reaction channel by redirecting fluid flowsdown those channels. Again, in those instances where the test compoundis immobilized on a bead, the immobilization will typically be via acleavable linker group, e.g., a photolabile, acid or base labile linkergroup. Accordingly, the test compound will typically need to be releasedfrom the bead, e.g., by exposure to a releasing agent such as light,acid, base or the like prior to flowing the test compound down thereaction channel.

[0109] Within the parallel channel, the test compound will be contactedwith the biochemical system for which an effector compound is beingsought. As shown, the first component of the biochemical system isplaced into the reaction channels using a similar technique to thatdescribed for the test compounds. In particular, the biochemical systemis typically introduced via one or more transverse seeding channels 306.Arrows 342 illustrate the direction of fluid flow within the seedingchannel 306. The biochemical system are optionally solution based, e.g.,a continuously flowing enzyme/substrate or receptor-ligand mixture, likethat described above, or as shown in FIGS. 4A-4F, may be a whole cell orbead based system, e.g., beads which have enzyme/substrate systemsimmobilized thereon.

[0110] In those instances where the biochemical system is incorporatedin a particle, e.g., a cell or bead, the parallel channel may include aparticle retention zone 344. Typically, such retention zones willinclude a particle sieving or filtration matrix, e.g., a porous gel ormicrostructure which retains particulate material but allows the freeflow of fluids. Examples of microstructures for this filtration include,e.g., those described in U.S. Pat. No. 5,304,487, which is herebyincorporated by reference in its entirety for all purposes. As with thecontinuous system, fluid direction within the more complex systems maybe generally controlled using microfabricated fluid directionstructures, e.g., pumps and valves. However, as the systems grow morecomplex, such systems become largely unmanageable. Accordingly,electroosmotic systems, as described above, are generally preferred forcontrolling fluid in these more complex systems. Typically, such systemswill incorporate electrodes within reservoirs disposed at the termini ofthe various transverse channels to control fluid flow thorough thedevice. In some aspects, it is desirable to include electrodes at thetermini of all the various channels. This generally provides for moredirect control, but also grows less manageable as systems grow morecomplex. In order to utilize fewer electrodes and thus reduce thepotential complexity, it may often be desirable in parallel systems,e.g., where two fluids are desired to move at similar rates in parallelchannels, to adjust the geometries of the various flow channels. Inparticular, as channel length increases, resistance along that channelwill also increase. As such, flow lengths between electrodes should bedesigned to be substantially the same regardless of the parallel pathchosen. This will generally prevent the generation of transverseelectrical fields and thus promote equal flow in all parallel channels.To accomplish substantially the same resistance between the electrodes,one can alter the geometry of the channel structure to provide for thesame channel length, and thus, the channel resistance, regardless of thepath travelled. Alternatively, resistance of channels are optionallyadjusted by varying the cross-sectional dimensions of the paths, therebycreating uniform resistance levels regardless of the path taken.

[0111] As the test compounds are drawn through their respective parallelreaction channels, they will contact the biochemical system in question.As described above, the particular biochemical system will typicallyinclude a flowable indicator system which indicates the relativefunctioning of that system, e.g., a soluble indicator such aschromogenic or fluorogenic substrate, labelled ligand, or the like, or aparticle based signal, such as a precipitate or bead bound signallinggroup. The flowable indicator is then flowed through the respectiveparallel channel and into the collection channel 308 whereupon thesignals from each of the parallel channels are flowed, in series, pastthe detection window, 116.

[0112] FIGS. 4A-4F, with reference to FIG. 3, show a schematicillustration of the progression of the injection of test compounds andbiochemical system components into the “serial input parallel reaction”device, exposure of the system to the test compounds, and flowing of theresulting signal out of the parallel reaction channels and past thedetection window. In particular, FIG. 4A shows the introduction of testcompounds immobilized on beads 346 through sample injection channel 304.Similarly, the biochemical system components 348 are introduced into thereaction channels 312-324 through seeding channel 306. Although shown asbeing introduced into the device along with the test compounds, asdescribed above, the components of the model system to be screened areoptionally incorporated into the reaction channels during manufacture.Again, such components are optionally provided in liquid form or inlyophilized form for increased shelf life of the particular screeningdevice.

[0113] As shown, the biochemical system components are embodied in acellular or particle based system, however, fluid components may also beused as described herein. As the particulate components flow into thereaction channels, they are optionally retained upon an optionalparticle retaining matrix 344, as described above.

[0114]FIG. 4B illustrates the release of test compounds from the beads346 by exposing the beads to a releasing agent. As shown, the beads areexposed to light from an appropriate light source 352, e.g., which isable to produce light in a wavelength sufficient to photolyze the linkergroup, thereby releasing compounds that are coupled to their respectivebeads via a photolabile linker group.

[0115] In FIG. 4C, the released test compounds are flowed into and alongthe parallel reaction channels as shown by arrows 354 until they contactthe biochemical system components. The biochemical system components 348are then allowed to perform their function, e.g., enzymatic reaction,receptor/ligand interaction, and the like, in the presence of the testcompounds. Where the various components of the biochemical system areimmobilized on a solid support, release of the components from theirsupports can provide the initiating event for the system. A solublesignal 356 which corresponds to the functioning of the biochemicalsystem is then generated (FIG. 4D). As described previously, a variationin the level of signal produced is an indication that the particulartest compound is an effector of the particular biochemical system. Thisis illustrated by the lighter shading of signal 358.

[0116] In FIGS. 4E and 4F, the soluble signal is then flowed out ofreactions channels 312-324 into the detection channel 308, and along thedetection channel past the detection window 116.

[0117] Again, a detection system as described above, located adjacentthe detection window will monitor the signal levels. In someembodiments, the beads which bore the test compounds are optionallyrecovered to identify the test compounds which were present thereon.This is typically accomplished by incorporation of a tagging groupduring the synthesis of the test compound on the bead. As shown, spentbead 360, i.e., from which a test compound has been released, isoptionally transported out of the channel structure through port 362 foridentification of the test compound that had been coupled to it. Suchidentification are optionally accomplished outside of the device bydirecting the bead to a fraction collector, whereupon the test compoundspresent on the beads are optionally identified, either throughidentification of a tagging group, or through identification of residualcompounds. Incorporation of tagging groups in combinatorial chemistrymethods has been previously described using encrypted nucleotidesequences or chlorinated/fluorinated aromatic compounds as tagginggroups. See, e.g., Published PCT Application No. WO 95/12608.Alternatively, the beads are optionally transported to a separate assaysystem within the device itself whereupon the identification is carriedout.

[0118]FIG. 6A shows an alternate embodiment of a “serial input parallelreaction” device which can be used for fluid based as opposed to beadbased systems. As shown the device 600 generally incorporates at leasttwo transverse channels as were shown in FIGS. 3 and 4, namely, sampleinjection channel 604 and detection channel 606. These transversechannels are interconnected by the series of parallel channels 612-620which connect sample channel 604 to detection channel 606.

[0119] The device shown also includes an additional set of channels fordirecting the flow of fluid test compounds into the reaction channels.In particular, an additional transverse pumping channel 634 is fluidlyconnected to sample channel 604 via a series of parallel pumpingchannels 636-646. The pumping channel includes reservoirs 650 and 652 atits termini. The intersections of parallel channels 636-646 arestaggered from the intersections of parallel channels 612-620 withsample channel 604, e.g., half way between. Similarly, transversepumping channel 608 is connected to detection channel 606 via parallelpumping channels 622-632. Again, the intersections of parallel pumpingchannels 622-632 with detection channel 606 are staggered from theintersections of reaction channels 612-620 with the detection channel606.

[0120] A schematic illustration of the operation of this system is shownin FIGS. 6B-6C. As shown, a series of test compounds, physicallyisolated from each other in separate subject material regions, areintroduced into sample channel 604 using the methods describedpreviously. For electroosmotic systems, potentials are applied at theterminus of sample channel 604, as well as reservoir 648. Potentials arealso applied at reservoirs 650:652, 654:656, and 658:660. This resultsin a fluid flow along the transverse channels 634, 604, 606 and 608, asillustrated by the arrows, and a zero net flow through the parallelchannel arrays interconnecting these transverse channels, as shown inFIG. 6B. Once the subject material regions containing the test compoundsare aligned with parallel reaction channels 612-620, connecting samplechannel 604 to detection channel 606, as shown by the shaded areas inFIG. 6B, flow is stopped in all transverse directions by removing thepotentials applied to the reservoirs at the ends of these channels. Asdescribed above, the geometry of the channels can be varied to maximizethe use of space on the substrate. For example, where the sample channelis straight, the distance between reaction channels (and thus, thenumber of parallel reactions that can be carried out in a size limitedsubstrate) is dictated by the distance between subject material regions.These restrictions, however, can be eliminated through the inclusion ofaltered channel geometries. For example, in some aspects, the length ofa first and second spacer regions can be accommodated by a serpentine,square-wave, saw tooth or other reciprocating channel geometry. Thisallows packing a maximum number of reaction channels onto the limitedarea of the substrate surface.

[0121] Once aligned with the parallel reaction channels, the sample, orsubject material, is then moved into the parallel reaction channels612-620 by applying a first potential to reservoirs 650 and 652, whileapplying a second potential to reservoirs 658 and 660, whereby fluidflow through parallel pumping channels 636-646 forces the subjectmaterial into parallel reaction channels 612-620, as shown in FIG. 6C.During this process, no potential is applied at reservoirs—648, 654,656, or the terminus of sample channel 604. Parallel channels 636-646and 622-632 are generally adjusted in length such that the total channellength, and thus the level of resistance, from reservoirs 650 and 652 tochannel 604 and from reservoirs 658 and 660 to channel 606, for any pathtaken will be the same. Resistance can generally be adjusted byadjusting channel length or width. For example, channels can belengthened by including folding or serpentine geometries. Although notshown as such, to accomplish this same channel length, channels 636 and646 would be the longest and 640 and 642 the shortest, to createsymmetric flow, thereby forcing the samples into the channels. As can beseen, during flowing of the samples through channels 612-620, theresistance within these channels will be the same, as the individualchannel length is the same.

[0122] Following the reaction to be screened, the subject materialregion/signal element is moved into detection channel 606 by applying apotential from reservoirs 650 and 652 to reservoirs 658 and 660, whilethe potentials at the remaining reservoirs are allowed to float. Thesubject material regions/signal are then serially moved past thedetection window/detector 662 by applying potentials to reservoirs 654and 656, while applying appropriate potentials at the termini of theother transverse channels to prevent any flow along the various parallelchannels.

[0123] Although shown with channels which intersect at right angles, itwill be appreciated that other geometries are also appropriate forserial input parallel reactions. For example, U.S. Ser. No. 08/835,101,filed Apr. 4, 1997, describes advantages to parabolic geometries andchannels which vary in width for control of fluid flow. In brief, fluidflow in electroosmotic systems is controlled by and therefore related tocurrent flow between electrodes. Resistance in the fluid channels variesas a function of path length and width, and thus, different lengthchannels have different resistances. If this differential in resistanceis not corrected for, it results in the creation of transverseelectrical fields which can inhibit the ability of the devices to directfluid flow to particular regions. The current, and thus the fluid flow,follows the path of least resistance, e.g., the shortest path. Whilethis problem of transverse electrical fields is alleviated through theuse of separate electrical systems, i.e., separate electrodes, at thetermini of each and every parallel channel, production of devicesincorporating all of these electrodes, and control systems forcontrolling the electrical potential applied at each of these electrodescan be complex, particularly where one is dealing with hundreds tothousands of parallel channels in a single small scale device, e.g., 1-2cm². Accordingly, the present invention provides microfluidic devicesfor affecting serial to parallel conversion, by ensuring that currentflow through each of a plurality of parallel channels is at anappropriate level to ensure a desired flow pattern through thosechannels or channel networks. A number of methods and substrate/channeldesigns for accomplishing these goals are appropriate.

[0124] In one example of parabolic geometry for the channels in anapparatus of the invention, the substrate includes a main channel. Aseries of parallel channels terminate in a main channel. The oppositetermini of these parallel channels are connected to parabolic channels.Electrodes are disposed at the termini of these parabolic channels. Thecurrent flow in each of the parallel channels is maintained constant orequivalent, by adjusting the length of the parallel channels, resultingin a parabolic channel structure connecting each of the parallelchannels to its respective electrodes. The voltage drop within theparabolic channel between the parallel channels is maintained constantby adjusting the channel width to accommodate variations in the channelcurrent resulting from the parallel current paths created by theseparallel channels. The parabolic design of the channels, in combinationwith their tapering structures, results in the resistance along all ofthe parallel channels being equal, resulting in an equal fluid flow,regardless of the path chosen. Generally, determining the dimensions ofchannels to ensure that the resistances among the channels arecontrolled as desired, may be carried out by well known methods, andgenerally depends upon factors such as the make-up of the fluids beingmoved through the substrates.

[0125] Although generally described in terms of screening assays foridentification of compounds which affect a particular interaction, basedupon the present disclosure, it will be readily appreciated that theabove described microlaboratory systems may also be used to screen forcompounds which specifically interact with a component of a biochemicalsystem without necessarily affecting an interaction between thatcomponent and another element of the biochemical system. Such compoundstypically include binding compounds which may generally be used in,e.g., diagnostic and therapeutic applications as targeting groups fortherapeutics or marker groups, i.e. radionuclides, dyes and the like.For example, these systems are optionally used to screen test compoundsfor the ability to bind to a given component of a biochemical system.

[0126] II. Microlaboratory System

[0127] Although generally described in terms of individual discretedevices, for ease of operation, the systems described will typically bea part of a larger system which can monitor and control the functioningof the devices, either on an individual basis, or in parallel,multi-device screens. An example of such a system is shown in FIG. 7.

[0128] As shown in FIG. 7, the system may include a test compoundprocessing system 700. The system shown includes a platform 702 whichcan hold a number of separate assay chips or devices 704. As shown, eachchip includes a number of discrete assay channels 706, each having aseparate interface 708, e.g., pipettor, for introducing test compoundsinto the device. These interfaces are used to sip test compounds intothe device, separated by sipping first and second spacer fluids, intothe device. In the system shown, the interfaces of the chip are insertedthrough an opening 710 in the bottom of the platform 702, which iscapable of being raised and lowered to place the interfaces in contactwith test compounds or wash/first spacer fluids/second spacer fluids,which are contained in, e.g., multiwell micro plates 711, positionedbelow the platform, e.g., on a conveyor system 712. In operation,multiwell plates containing large numbers of different test compoundsare stacked 714 at one end of the conveyor system. The plates are placedupon the conveyor separated by appropriate buffer reservoirs 716 and718, which may be filled by buffer system 720. The plates are steppeddown the conveyor and the test compounds are sampled into the chips,interspersed by appropriate spacer fluid regions. After loading the testcompounds into the chips, the multiwell plates are then collected orstacked 722 at the opposite end of the system. The overall controlsystem includes a number of individual microlaboratory systems ordevices, e.g., as shown in FIG. 7. Each device is connected to acomputer system which is appropriately programmed to control fluid flowand direction within the various chips, and to monitor, record andanalyze data resulting from the screening assays that are performed bythe various devices. The devices will typically be connected to thecomputer through an intermediate adapter module which provides aninterface between the computer and the individual devices forimplementing operational instructions from the computer to the devices,and for reporting data from the devices to the computer. For example,the adapter will generally include appropriate connections tocorresponding elements on each device, e.g., electrical leads connectedto the reservoir based electrodes that are used for electroosmotic fluidflow, power inputs and data outputs for detection systems, eitherelectrical or fiberoptic, and data relays for other sensor elementsincorporated into the devices. The adapter device may also provideenvironmental control over the individual devices where such control isnecessary, e.g., maintaining the individual devices at optimaltemperatures for performing the particular screening assays.

[0129] As shown, each device is also equipped with appropriate fluidinterfaces, e.g., micropipettors, for introducing test compounds intothe individual devices. The devices may readily be attached to roboticsystems which allow test compounds to be sampled from a number ofmultiwell plates that are moved along a conveyor system. Interveningspacer fluid regions can also be introduced via a spacer solutionreservoir.

[0130] III. Fluid Electrode Interface to Prevent Degradation of ChemicalSpecies in a Microchip

[0131] When pumping fluids or other materials electroosmotically orelectrophoretically through an apparatus of the invention, chemicalspecies in the fluid can be degraded if high voltages or currents areapplied, or if voltages are applied for a long period of time. Designswhich retard movement of chemical species from the electrode to achannel entrance or retard the movement of chemical species to theelectrode improve performance of chemical assays by reducing unwanteddegradation of chemical species within the sample. These designs areparticularly preferred in assay systems where voltages are applied forlong periods, e.g., several hours to several days.

[0132] Electrode designs which reduce degradation of chemical species inthe assays of the invention are illustrated by consideration of FIG. 12,panels A-G. The designs retard the moving of chemical species from theelectrode to the channel entrance or retard the movement of chemicalspecies to the electrode improve performance of chemical assays. FIG.12A shows a typical electrode design, in which electrode 1211 ispartially submerged in reservoir 1215 fluidly connected to fluid channel1217.

[0133] In comparison, FIG. 12B utilizes a salt bridge between electrodewith frit 1219 and fluid reservoir 1221 fluidly connected to fluidchannel 1223.

[0134]FIG. 12C reduces degradation of chemical species by providingelectrode 1225 submersed in first fluid reservoir 1227 fluidly connectedto second fluid reservoir 1229 by large channel 1231 which limitsdiffusion, but has a low electroosmotic flow.

[0135]FIG. 12D provides a similar two part reservoir, in which electrode1235 is submersed in first fluid reservoir 1237 fluidly connected tosecond fluid reservoir 1241 by small channel 1243 which is treated toreduce or eliminate electroosmotic flow.

[0136]FIG. 12E provides another similar two part reservoir, in whichelectrode 1245 is submersed in first fluid reservoir 1247 fluidlyconnected to second fluid reservoir 1251 by channel 1253. Channel 1253is filled with a material such as gel, Agar, glass beads or other matrixmaterial for reducing electroosmotic flow.

[0137]FIG. 12F provides a variant two part reservoir system, in whichelectrode 1255 is submersed in first fluid reservoir 1257 fluidlyconnected to second fluid reservoir 1259 by channel 1261. The fluidlevel in second fluid reservoir 1259 is higher than the fluid level infirst fluid reservoir 1257, which forces fluid towards electrode 1255.

[0138]FIG. 12G provides a second variant two part reservoir, in whichelectrode 1265 is submersed in first fluid reservoir 1267 fluidlyconnected to second fluid reservoir 1269 by channel 1271. The diameteron first fluid reservoir 1267 is small enough that capillary forces drawfluid into first fluid reservoir 1267.

[0139] Modifications can be made to the method and apparatus ashereinbefore described without departing from the spirit or scope of theinvention as claimed, and the invention can be put to a number ofdifferent uses, including:

[0140] The use of a microfluidic system containing at least a firstsubstrate having a first channel and a second channel intersecting saidfirst channel, at least one of said channels having at least onecross-sectional dimension in a range from 0.1 to 500 μm, in order totest the effect of each of a plurality of test compounds on abiochemical system.

[0141] The use of a microfluidic system as hereinbefore described,wherein said biochemical system flows through one of said channelssubstantially continuously, enabling sequential testing of saidplurality of test compounds.

[0142] The use of a microfluidic system as hereinbefore described,wherein the provision of a plurality of reaction channels in said firstsubstrate enables parallel exposure of a plurality of test compounds toat least one biochemical system.

[0143] The use of a microfluidic system as hereinbefore described,wherein each test compound is physically isolated from adjacent testcompounds.

[0144] The use of a substrate carrying intersecting channels inscreening test materials for effect on a biochemical system by flowingsaid test materials and biochemical system together using said channels.

[0145] The use of a substrate as hereinbefore described, wherein atleast one of said channels has at least one cross-sectional dimension ofrange 0.1 to 500 μm.

[0146] An assay utilizing a use of any one of the microfluidic systemsor substrates hereinbefore described.

[0147] The invention provides, inter alia, an apparatus for detecting aneffect of a test compound on a biochemical system, comprising asubstrate having at least one surface with a plurality of reactionchannels fabricated into the surface. Apparatus as hereinbeforedescribed, having at least two transverse channels fabricated into thesurface, wherein each of the plurality of reaction channels is fluidlyconnected to a first of the at least two transverse channels at a firstpoint in each of the reaction channels, and fluidly connected to asecond transverse channel at a second point in each of the reactionchannels and an assay apparatus including an apparatus as hereinbeforedescribed are also provided.

EXAMPLES

[0148] The following examples are provided by way of illustration onlyand not by way of limitation. Those of skill will readily recognize avariety of noncritical parameters which can be changed or modified toyield essentially similar results.

Example 1 Enzyme Inhibitor Screen

[0149] The efficacy of performing an enzyme inhibition assay screen wasdemonstrated in a planar chip format. A 6-port planar chip was employedhaving the layout shown in FIG. 8. The numbers adjacent the channelsrepresent the lengths of each channel in millimeters. Two voltage stateswere applied to the ports of the chip. The first state (State 1)resulted in flowing of enzyme with buffer from the top buffer well intothe main channel. The second voltage state (State 2) resulted in theinterruption of the flow of buffer from the top well, and theintroduction of inhibitor from the inhibitor well, into the main channelalong with the enzyme. A control experiment was also run in which bufferwas placed into the inhibitor well.

[0150] Applied voltages at each port for each of the two applied voltagestates were as follows: State 1 State 2 Top Buffer Well (I) 1831 1498Inhibitor Well (II) 1498 1900 Enzyme Well (III) 1891 1891 Substrate Well(IV) 1442 1442 Bottom Buffer Well (V) 1442 1442 Detect./Waste Well (VI)0 0

[0151] To demonstrate the efficacy of the system, an assay was designedto screen inhibitors of β-galactosidase using the followingenzyme/substrate/inhibitor reagents: Enzyme: β-Galactosidase (180 U/mlin 50 mM Tris/300 μg/ml BSA Substrate: Fluorescein-digalactoside (FDG)400 μM Inhibitor: IPTG, 200 mM Buffer: 20 mM Tris, pH 8.5

[0152] Enzyme and substrate were continually pumped through the mainchannel from their respective ports under both voltage states. Inhibitoror Buffer were delivered into the main channel alternately from theirrespective wells by alternating between voltage state 1 and voltagestate 2. When no inhibitor was present at the detection end of the mainchannel, a base line level of fluorescent product was produced. Uponintroduction of inhibitor, the fluorescent signal was greatly reduced,indicating inhibition of the enzyme/substrate interaction. Fluorescentdata obtained from the alternating delivery of inhibitor and buffer intothe main channel is shown in FIG. 9A. FIG. 9B a superposition of the twodata segments from FIG. 9A, directly comparing the inhibitor data withcontrol (buffer) data. The control shows only a minor fluctuation in thefluorescent signal that apparently resulted from a dilution of theenzyme substrate mixture, whereas the inhibitor screen shows asubstantial reduction in the fluorescent signal, indicating clearinhibition.

Example 2 Screening of Multiple Test Compounds

[0153] An assay screen is performed to identify inhibitors of anenzymatic reaction. A schematic of the chip to be used is shown in FIG.10. The chip has a reaction channel 5 cm in length which includes a 1 cmincubation zone and a 4 cm reaction zone. The reservoir at the beginningof the sample channel is filled with enzyme solution and the sidereservoir is filled with the fluorogenic substrate. Each of the enzymeand substrate are diluted to provide for a steady state signal in thelinear signal range for the assay system, at the detector. Potentialsare applied at each of the reservoirs (sample source, enzyme, substrateand waste) to achieve an applied field of 200 V/cm. This applied fieldproduces a flow rate of 2 mm/second. During passage of a given samplethrough the chip, there will generally be a diffusive broadening of thesample. For example, in the case of a small molecule sample, e.g., 1 mMbenzoic acid diffusive broadening of approximately 0.38 mm and anelectrophoretic shift of 0.4 mm is seen.

[0154] Subject material regions containing test compounds in 150 mM NaClare introduced into the sample channel separated by first spacer regionsof 150 mM NaCl and second spacer regions of 5 mM borate buffer. Onceintroduced into the sample channel shown, the subject material regionrequires 12 seconds to travel the length of the sample channel and reachthe incubation zone of the reaction channel. This is a result of theflow rate of 2 mm/sec, allowing for 1 second for moving the samplepipettor from the sample to the spacer compounds. Allowing for theseinterruptions, the net flow rate is 0.68 mm/sec. Another 12 seconds isrequired for the enzyme/test compound mixture to travel through theincubation zone to the intersection with the substrate channel wheresubstrate is continuously flowing into the reaction zone of the reactionchannel. Each subject material region containing the test compounds thenrequires 48 seconds to travel the length of the reaction zone and pastthe fluorescence detector. A schematic of timing for subject materialregion/spacer region loading is shown in FIG. 11. The top panel showsthe subject material/first spacer region/second spacer regiondistribution within a channel, whereas the lower panel shows the timingrequired for loading the channel. As shown, the schematic includes theloading (sipping) of high salt (HS) first spacer fluid (“A”), moving thepipettor to the sample or subject material (“B”), sipping the sample orsubject material (“C”), moving the pipettor to the high salt firstspacer fluid (“D”) sipping the first spacer fluid (“E”), moving thepipettor to the low salt (LS) or second spacer fluid (“F”), sipping thesecond spacer fluid (“G”) and returning to the first spacer fluid (“H”).The process is then repeated for each additional test compound.

[0155] A constant base fluorescent signal is established at the detectorin the absence of test compounds. Upon introduction of the testcompounds, a decrease in fluorescence is seen similar to that shown inFIGS. 9A and 9B, which, based upon time delays, corresponds to aspecific individual test compound. This test compound is tentativelyidentified as an inhibitor of the enzyme, and further testing isconducted to confirm this and quantitate the efficacy of this inhibitor.

[0156] While the foregoing invention has been described in some detailfor purposes of clarity and understanding, it will be clear to oneskilled in the art from a reading of this disclosure that variouschanges in form and detail can be made without departing from the truescope of the invention. All publications and patent documents cited inthis application are incorporated by reference in their entirety for allpurposes to the same extent as if each individual publication or patentdocument were so individually denoted.

What is claimed is:
 1. An apparatus for screening test compounds for an effect on a biochemical system, comprising: a substrate having at least one surface; at least two intersecting channels fabricated into said surface of said substrate, at least one of said at least two intersecting channels having at least one cross-sectional dimension in the range from about 0.1 to about 500 μm; a source of a plurality of different test compounds fluidly connected to a first of said at least two intersecting channels; a source of at least one component of said biochemical system fluidly connected to a second of said at least two intersecting channels; a fluid direction system for flowing said at least one component within said second of said at least two intersecting channels and for introducing said different test compounds from said first to said second of said at least two intersecting channels; a cover mated with said surface; and a detection zone in said second channel for detecting an effect of said test compound on said biochemical system.
 2. The apparatus of claim 1, wherein said fluid direction system generates a continuous flow of said at least first component along said second of said at least two intersecting channels, and periodically injects a test compound from said first channel into said second channel.
 3. The apparatus of claim 1, further comprising a source of a second component of said biochemical system, and a third channel fabricated into said surface, said third channel fluidly connecting at least one of said at least two intersecting channels with said source of said second component of said biochemical system.
 4. The apparatus of claim 3, wherein said fluid direction system generates a continuous flow of a mixture of said first component and said second component along said second of said at least two intersecting channels, and periodically injects a test compound from said first channel into said second channel.
 5. The apparatus of claim 1, wherein said fluid direction system continuously flows said plurality of different test compounds from said first into said second of said at least two intersecting channels, each of said plurality of different test compounds being separated by a fluid spacer.
 6. The apparatus of claim 1, wherein said fluid direction system comprises: at least three electrodes, each electrode being in electrical contact with said at least two intersecting channels on a different side of an intersection formed by said at least two intersecting channels; and a control system for concomitantly applying a variable voltage at each of said electrodes, whereby movement of said test compounds or said at least first component in said at least two intersecting channels are controlled.
 7. The apparatus of claim 1, wherein said detection system includes a detection window in said second channel.
 8. The apparatus of claim 7, wherein said detection system is a fluorescent detection system.
 9. The apparatus of claim 1, wherein said substrate is planar.
 10. The apparatus of claim 1, wherein said substrate comprises etched glass.
 11. The apparatus of claim 1, wherein said substrate comprises etched silicon.
 12. The apparatus of claim 1, further comprising an insulating layer disposed over said etched silicon substrate.
 13. The apparatus of claim 1, wherein said substrate is a molded polymer.
 14. The apparatus of claim 1, wherein said at least one component of a biochemical system comprises an enzyme, and a substrate which produces a detectable signal when reacted with said enzyme.
 15. The apparatus of claim 14, wherein said substrate is selected from the group consisting of chromogenic and fluorogenic substrates.
 16. The apparatus of claim 1, wherein said at least first component of a biochemical system comprises a receptor/ligand binding pair, wherein at least one of said receptor or ligand has a detectable signal associated therewith.
 17. The apparatus of claim 1, wherein said first component of a biochemical system comprises a receptor/ligand binding pair, wherein binding of said receptor to said ligand produces a detectable signal.
 18. The apparatus of claim 1, the apparatus further comprising a plurality of electrodes in a plurality of reservoirs fluidly connected to one or more of said intersecting channels and a control system for concomitantly applying a voltage to each of said electrodes, whereby movement of said first component in said at least two intersecting channels is controlled.
 19. The apparatus of claim 18, wherein the apparatus minimizes degradation of chemical species present in said reservoirs or said intersecting channels.
 20. The apparatus of claim 19, wherein the apparatus further comprises one or more component for reducing electroosmotic flow, selected from the group consisting of: a frit on one or more of the electrodes, which frit reduces electroosmotic flow towards the one or more electrodes; a large channel between at least two of said reservoirs, which large channel limits diffusion of said chemical species, the channel having low electroosmotic flow; a narrow channel between at least two of said reservoirs, which narrow channel limits diffusion of said chemical species, the narrow channel treated to reduce electroosmotic flow; a filled channel between at least two of said reservoirs, which filled channel comprises a matrix to limit transport of said chemical species through the filled channel, the filled channel thereby having low electroosmotic flow; a high reservoir having a fluid level higher than at least one low reservoir, which high reservoir is fluidly connected to said low reservoir, which low reservoir comprises an electrode, wherein fluid pressure between the high reservoir and the low reservoir reduces electroosmotic flow towards the electrode; and, a dual reservoir system with a first reservoir fluidly connected through a connecting channel to a narrow diameter second reservoir adapted to receive one of the plurality of electrodes, said narrow diameter second reservoir adapted to draw fluid by capillary electrophoresis towards the one electrode, thereby countering electroosmotic flow in the connecting channel.
 21. An apparatus for detecting an effect of a test compound on a biochemical system, comprising: a substrate having at least one surface; a plurality of reaction channels fabricated into said surface; at least two transverse channels fabricated into said surface, each of said plurality of reaction channels being fluidly connected to a first of said at least two transverse channels at a first point in said reaction channels, and fluidly connected to a second of said at least two transverse channels at a second point in said reaction channels, said at least two transverse channels and said plurality of reaction channels each having at least one cross-sectional dimension in the range from about 0.1 to about 500 μm; a source of at least one component of said biochemical system, said source of at least one component of said biochemical system being fluidly connected to each of said plurality of reaction channels; a source of test compounds fluidly connected to said first of said at least two transverse channels; a fluid direction system for controlling movement of said test compound and said at least one component within said at least two transverse channels and said plurality of reaction channels; a cover mated with said surface; and a detection system for detecting an effect of said test compound on said biochemical system.
 22. The apparatus of claim 21, wherein said fluid control system comprises: a plurality of individual electrodes, each in electrical contact with each terminus of said at least two transverse channels; and a control system for concomitantly applying a variable voltage at each of said electrodes, whereby movement of said test compounds or said at least first component in said at least two transverse channels and said plurality of reaction channels are controlled.
 23. The apparatus of claim 21, wherein each of said plurality of reaction channels comprises a bead resting well at said first point in said plurality of reaction channels.
 24. The apparatus of claim 21, wherein said source of at least one component of a biochemical system is fluidly connected to said plurality of reaction channels by a third transverse channel, said third transverse channel having at least one cross sectional dimension in a range of from 0.1 to 500 μm and being fluidly connected to each of said plurality of reaction channels at a third point in said reaction channels.
 25. The apparatus of claim 21, wherein said third point in said reaction channels is intermediate to said first and second points in said reaction channels.
 26. The apparatus of claim 25, further comprising a particle retention zone in each of said plurality of reaction channels, between said third and said second points in said plurality of reaction channels.
 27. The apparatus of claim 26, wherein said particle retention zone comprises a particle retention matrix.
 28. The apparatus of claim 26, wherein said particle retention zone comprises a microstructural filter.
 29. The apparatus of claim 21, wherein said plurality of reaction channels comprises a plurality of parallel reaction channels fabricated into said surface of said substrate and said at least two transverse channels are connected at opposite ends of each of said parallel reaction channels.
 30. The apparatus of claim 21, wherein said at least two transverse channels are fabricated on said surface of said substrate in inner and outer concentric channels, respectively, and said plurality of reaction channels extend radially from said inner concentric channel to said outer concentric channel.
 31. The apparatus of claim 30, wherein said detection system comprises a detection window in said second channel.
 32. The apparatus of claim 30, wherein said detection system is a fluorescent detection system.
 33. The apparatus of claim 21, wherein said substrate is planar.
 34. The apparatus of claim 21, wherein said substrate comprises etched glass.
 35. The apparatus of claim 21, wherein said substrate comprises etched silicon.
 36. The apparatus of claim 21, further comprising an insulating layer disposed over said etched silicon substrate.
 37. The apparatus of claim 21, wherein said substrate is a molded polymer.
 38. The apparatus of claim 21, wherein said at least one component of a biochemical system comprises an enzyme, and an enzyme substrate which produces a detectable signal when reacted with said enzyme.
 39. The apparatus of claim 38, wherein said enzyme substrate is selected from the group consisting of chromogenic and fluorogenic substrates.
 40. The apparatus of claim 21, wherein said at least first component of a biochemical system comprises a receptor/ligand binding pair, wherein at least one of said receptor or ligand has a detectable signal associated therewith.
 41. The apparatus of claim 21, wherein said first component of a biochemical system comprises a receptor/ligand binding pair, wherein binding of said receptor to said ligand produces a detectable signal.
 42. A method of determining whether a sample contains a compound capable of affecting a biochemical system, comprising: providing a substrate having at least a first surface, and at least two intersecting channels fabricated in said first surface, at least one of said at least two intersecting channels having at least one cross-sectional dimension in a range from 0.1 to 500 μm; flowing a first component of a biochemical system in a first of said at least two intersecting channels; flowing said sample from a second channel into said first channel whereby said sample contacts said first component of said biochemical system; and detecting an effect of said at least sample on said biochemical system.
 43. The method of claim 42, wherein said at least first component of a biochemical system comprises at least one member of an antibody/antigen binding pair, wherein said antibody is specifically immunoreactive with said antigen.
 44. The method of claim 42, wherein said at least first component of a biochemical system comprises an antibody and an antigen specifically reactive with said antibody.
 45. The method of claim 42, wherein one of said antibody or antigen comprises a detectable labelling group.
 46. The method of claim 42, wherein said at least first component of a biochemical system comprises at least one member of a receptor/ligand binding pair.
 47. The method of claim 42, wherein said at least first component of a biochemical system comprises a receptor and a ligand capable of specifically binding to said ligand.
 48. The method of claim 42, wherein said sample is derived from a patient.
 49. The method of claim 48, wherein said sample is blood-derived.
 50. The method of claim 42, wherein said detecting step comprises measuring a parameter of said biochemical system in the presence and absence of said sample, and comparing the measured parameter in the presence of said sample to the measured parameter in the absence of said sample, a change in said parameter being indicative that said sample has an effect on said biochemical system.
 51. An apparatus for screening test compounds for an effect on a biochemical system, comprising: a substrate having at least one surface, and comprising at least two intersecting channels fabricated into said surface of said substrate, at least one of said at least two intersecting channels having at least one cross-sectional dimension in the range from about 0.1 to about 500 μm; a source of a sample fluidly connected to a first of said at least two intersecting channels; a source of at least one component of said biochemical system fluidly connected to a second of said at least two intersecting channels; a fluid direction system for flowing said at least one component within said second of said at least two intersecting channels and for introducing said sample from said first to said second of said at least two intersecting channels; a cover mated with said surface; and a detection zone in said second channel for detecting an effect of said sample on said biochemical system.
 52. A method of screening a plurality of test compounds for an effect on a biochemical system, comprising: providing a substrate having at least a first surface, and at least two intersecting channels fabricated in said first surface, at least one of said at least two intersecting channels having at least one cross-sectional dimension in a range from 0.1 to 500 μm; flowing a first component of a biochemical system in a first of said at least two intersecting channels; flowing at least a first test compound from a second channel into said first channel whereby said first test compound contacts said first component of said biochemical system; and detecting an effect of said at least first test compound on said biochemical system.
 53. The method of claim 52, wherein said at least first component of a biochemical system produces a detectable signal representative of a function of said biochemical system.
 54. The method of claim 52, wherein said at least first component further comprises an indicator compound which interacts with said first component to produce a detectable signal representative of a functioning of said biochemical system.
 55. The method of claim 52, wherein said first component of a biochemical system comprises an enzyme and a substrate for said enzyme, wherein action of said enzyme on said substrate produces a detectable signal.
 56. The method of claim 52, wherein said first component of a biochemical system comprises a receptor/ligand binding pair, wherein at least one of said receptor or ligand has a detectable signal associated therewith.
 57. The method of claim 52, wherein said first component of a biochemical system comprises a receptor/ligand binding pair, wherein binding of said receptor to said ligand produces a detectable signal.
 58. The method of claim 52, wherein said at least first component of a biochemical system is a biological barrier and said effect of said at least first test compound is an ability of said test compound to traverse said barrier.
 59. The method of claim 58, wherein said barrier is selected from the group consisting of an epithelial or an endothelial layer.
 60. The method of claim 52, wherein said at least first component of a biochemical system comprises cells, and said detecting step comprises determining an effect of said test compound on said cells.
 61. The method of claim 60, wherein said cells are capable of producing a detectable signal corresponding to a cellular function, and said detecting step comprises detecting an effect of said test compound on said cellular function by detecting a level of said detectable signal.
 62. The method of claim 60, wherein said detecting step comprises detecting an effect of said test compound on viability of said cells.
 63. A method of screening a plurality of test compounds for an effect on a biochemical system, comprising: providing a substrate having at least a first surface, and at least two intersecting channels fabricated in said first surface, at least one of said at least two intersecting channels having at least one cross-sectional dimension in a range from 0.1 to 500 μm; continuously flowing a first component of a biochemical system in a first channel of said at least two intersecting channels; periodically introducing a different test compound into said first channel from a second channel of said at least two intersecting channels; and detecting an effect of said test compound on said at least first component of a biochemical system.
 64. The method of claim 63, wherein said step of periodically introducing comprises flowing a plurality of different test compounds into said first channel from a second channel of said at least two intersecting channels, each of said plurality of different test compounds being physically isolated from each other of said plurality of different test compounds.
 65. The method of claim 63, wherein said at least first component of a biochemical system produces a detectable signal representative of a function of said biochemical system.
 66. The method of claim 65, wherein said detecting comprises monitoring said detectable signal from said continuously flowing first component at a point on said first channel, said detectable signal having a steady state intensity, and wherein said effect of said interaction between said first component and said test compound comprises a deviation from said steady state intensity of said detectable signal.
 67. The method of claim 65, wherein said at least first component further comprises an indicator compound which interacts with said first component to produce a detectable signal representative of a functioning of said biochemical system.
 68. The method of claim 67, wherein said first component of a biochemical system comprises an enzyme and said indicator compound comprises a substrate for said enzyme, wherein action of said enzyme on said substrate produces a detectable signal.
 69. The method of claim 65, wherein said at least first component of a biochemical system comprises a receptor/ligand binding pair, wherein at least one of said receptor or ligand has a detectable signal associated therewith.
 70. The method of claim 69, wherein said receptor and said ligand flow along said first channel at different rates.
 71. The method of claim 65, wherein said first component of a biochemical system comprises a receptor/ligand binding pair, wherein binding of said receptor to said ligand produces a detectable signal.
 72. The method of claim 63, wherein said at least first component of a biochemical system comprises cells, and said detecting step comprises determining an effect of said test compound on said cells.
 73. The method of claim 72, wherein said cells are capable of producing a detectable signal corresponding to a cellular function, and said detecting step comprises detecting an effect of said test compound on said cellular function by detecting a level of said detectable signal.
 74. The method of claim 72, wherein said detecting step comprises detecting an effect of said test compound on viability of said cells.
 75. A method of screening a plurality of different test compounds for an effect on a biochemical system, comprising: providing a substrate having at least a first surface, and a plurality of reaction channels fabricated in said first surface, each of said plurality of reaction channels being fluidly connected to at least two transverse channels fabricated in said surface; introducing at least a first component of a biochemical system into said plurality of reaction channels; flowing a plurality of different test compounds through at least one of said at least two transverse channels, each of said plurality of test compounds being introduced into said at least one transverse channels in a separate subject material region; directing each of said plurality of different test compounds into a separate one of said plurality of reaction channels; and detecting an effect of each of said test compounds on said at least one component of said biochemical system.
 76. The method of claim 75, wherein said at least first component of said biochemical system produces a flowable detectable signal representative of a function of said biochemical system.
 77. The method of claim 76, wherein said detectable flowable signal produced in each of said plurality of reaction channels is flowed into and through said second transverse channel, each of said detectable flowable signals produced in each of said plurality of reaction channels being physically isolated from each other of said detectable flowable signals, whereupon each of said detectable flowable signals is separately detected.
 78. The method of claim 76, wherein said flowable signal comprises a soluble signal.
 79. The method of claim 78, wherein said soluble signal is selected from fluorescent or colorimetric signals.
 80. The method of claim 75, wherein said at least first component further comprises an indicator compound which interacts with said first component to produce a detectable signal representative of a functioning of said biochemical system.
 81. The method of claim 80, wherein said first component of a biochemical system comprises an enzyme and said indicator compound comprises a substrate for said enzyme, wherein action of said enzyme on said substrate produces a detectable signal.
 82. The method of claim 75, wherein said at least first component of a biochemical system comprises a receptor/ligand binding pair, wherein at least one of said receptor or ligand has a detectable signal associated therewith.
 83. The method of claim 75, wherein said first component of a biochemical system comprises a receptor/ligand binding pair, wherein binding of said receptor to said ligand produces a detectable signal.
 84. The method of claim 75, wherein said at least first component of a biochemical system comprises cells, and said detecting step comprises determining an effect of said test compound on said cells.
 85. The method of claim 84, wherein said cells are capable of producing a detectable signal corresponding to a cellular function, and said detecting step comprises detecting an effect of said test compound on said cellular function by detecting a level of said detectable signal.
 86. The method of claim 85, wherein said detecting step comprises detecting an effect of said test compound on viability of said cells.
 87. The method of claim 75, wherein each of said plurality of different test compounds is immobilized upon a separate bead, and said step of directing each of said plurality of different test compounds into a separate one of said plurality of reaction channels comprises: lodging one of said separate beads at an intersection of said first transverse channel and each of said plurality of reaction channels; and controllably releasing said test compounds from each of said separate beads into each of said plurality of reaction channels.
 88. The use of a microfluidic system containing at least a first substrate having a first channel and a second channel intersecting said first channel, at least one of said channels having at least one cross-sectional dimension in a range from 0.1 to 500 μm, in order to test the effect of each of a plurality of test compounds on a biochemical system.
 89. A use of claim 88, wherein said biochemical system flows through one of said channels substantially continuously, enabling sequential testing of said plurality of test compounds.
 90. A use of claim 88, or claim 89, wherein the provision of a plurality of reaction channels in said first substrate enables parallel exposure of a plurality of test compounds to at least one biochemical system.
 91. A use of any of claims 88, 89, or 90 wherein each test compound is physically isolated from adjacent test compounds.
 92. The use of a substrate carrying intersecting channels in screening test materials for effect on a biochemical system by flowing said test materials and biochemical system together using said channels.
 93. A use of claim 92, wherein at least one of said channels has at least one cross-sectional dimension of range 0.1 to 500 μm.
 94. An assay utilizing a use of any one of claims 88 to
 93. 95. An apparatus for detecting an effect of a test compound on a biochemical system, comprising a substrate having at least one surface with a plurality of reaction channels fabricated into the surface.
 96. An apparatus as claimed in claim 95, having at least two transverse channels fabricated into the surface, wherein each of the plurality of reaction channels is fluidly connected to a first of the at least two transverse channels at a first point in each of the reaction channels, and fluidly connected to a second transverse channel at a second point in each of the reaction channels.
 97. Assay apparatus including an apparatus as claimed in claim 95 or claim
 1. 